Compositions and Methods for Prognosis, Diagnosis, Prevention and Treatment of Cancers

ABSTRACT

The present invention provides compositions and methods of using the EPHB2 gene or its related signaling pathways to detect, prognosticate, assess the risk of, prevent, or treat cancers. Cancers amenable to the present invention include, but are not limited to, prostate cancer, breast cancer, and neuroblastoma. In one aspect, the present invention provides compositions which comprise an agent capable of eradicating or alleviating an abnormality in the EPHB2 gene or its related signaling pathways. This abnormality may cause or contribute to the development or progression of cancers. In another aspect, the present invention provides methods comprising detecting an abnormality in the EPHB2 gene or its related signaling pathways. The presence or absence of such an abnormality is indicative of the risk or disease status of cancer in a person of interest.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Nonprovisionalapplication Ser. No. 11/065,139, filed Feb. 24, 2005 and claims thebenefit of U.S. Provisional Application No. 60/546,949, filed Feb. 24,2004, and U.S. Provisional Application No. 60/599,062, filed Aug. 6,2004, all of which are incorporated herein by reference in theirentireties.

TECHNICAL FIELD

The present invention relates to compositions and methods of using theEPHB2 gene or its associated signaling pathways for the diagnosis,prognosis, prevention, or treatment of cancers.

BACKGROUND

Inactivation of tumor-suppression genes (TSGs) in cancer is often atwo-step process, involving a mutation of the target gene and additionalloss of the wild type allele. Mapping of chromosomal deletions andlosses of heterozygosity in cancer cells has been widely applied toguide the identification of TSGs. However, this approach alone is slow,labor intensive and complicated by genomic instability, often leading tonumerous candidate regions to study. In an alternative approach, thenonsense-mediated RNA decay (NMD) mechanism, which normally targetstranscripts with nonsense mutations for rapid degradation, can beblocked to cause differential stabilization of genes that harbortruncating mutations. By using microarrays to measure transcript levelsfollowing NMD inhibition, this approach has recently been proposed forgenome-wide identification of mutated genes in cell lines.

SUMMARY OF THE INVENTION

The present invention employs nonsense-mediated RNA decay microarraysand array-based comparative genomic hybridization (CGH) for theidentification of genes whose inactivation or deregulation leads to thedevelopment or progression of cancers. Using this approach, the receptortyrosine kinase gene EPHB2 was identified as a tumor suppressor gene.Inactivation or deregulation of this gene is associated with a varietyof cancers including prostate cancer, breast cancer, colon cancer, andneuroblastoma. Transfection of cancer cells, which lack functionalEphB2, with wild type EPHB2 suppresses clonogenic growth. The EPHB2 genecan therefore be used as a biological marker for the diagnosis,prognosis, or assessment of the risk or progression of cancers.Variations in the EPHB2 gene can also be used for discovering anddeveloping cancer drugs and have utility as makers for making medicaldecisions for the treatment or prevention of cancers, as well as beingused directly to pharmacologically treat cancer.

In one aspect, the present invention provides methods for the prognosisof a cancer in a patient of interest. These methods comprise detectingthe presence or absence of an abnormality in a biological sample of thepatient of interest. The presence of the abnormality is indicative of apoor prognosis of the patient of interest, and the absence of theabnormality is suggestive of a good prognosis of the patient ofinterest. Abnormalities that are suitable for cancer prognosis include,but are not limited to, mutations in the EPHB2 gene, inactivation of oneor more alleles of the EPHB2 gene, deletion of one or more alleles ofthe EPHB2 gene, a reduced level of expression of the EPHB2 gene, areduced level of activity of a EphB2 protein, a change in the normalfunction of EPHB2, or any combination thereof. Other abnormalities, suchas imbalances or deregulations in EphB2-mediated signal transductionpathways, can also be used for cancer prognosis. In some instances, theeffect of these imbalances or deregulations is similar to that ofbi-allelic inactivation of the EPHB2 gene. In one example, theseimbalances or deregulations can be corrected or alleviated byintroducing a wild type EphB2 protein or a functional component of thepathways into the affected cells.

Cancers that are amenable to the present invention include, but are notlimited to, prostate cancer, breast cancer, colon cancer, neuroblastoma,or other solid tumors that harbor EPHB2 gene mutations or deregulations.Leukemia, lymphoma, multiple myeloma, or other blood cancers can also beprognosticated according to the present invention. The extent orseverity of the mutation(s) in the EPHB2 gene or the deregulation of anEphB2-associated signal transductions pathway is correlated with theprogression or prognosis of the cancer. As a result, inactivation orderegulation of two alleles of the EPHB2 gene often indicates that thecancer is more difficult to treat than a cancer in which only one alleleof the EPHB2 is inactivated or deregulated. Likewise, a cancer havingone allele of the EPHB2 gene inactivated or deregulated is moredifficult to treat than a cancer in which both alleles of the EPHB2 geneare functional.

Many types of biological samples can be used for the prognosis of acancer in a patient of interest. In one example, the biological sampleused is prepared from the primary site of the cancer. A sample thusprepared comprises primary tumor cells. Samples isolated from metastaticsites of the cancer can also be used. These samples comprise metastatictumor cells. Abnormalities observed in either type of these cancer cellscan be used for the prognosis of a cancer in a patient of interest.Non-cancerous samples can also be used, such as blood samples or samplesisolated from potential metastatic sites. These samples can indicatesusceptibility of the patient to the invasion or spread of cancer cellsand, therefore, the chance of metastasis or prognosis of the patient.

In one embodiment, the prognosis of a cancer in a patient of interest isevaluated by detecting the presence or absence of a mutation ormutations in the EPHB2 gene in a biological sample of the patient (e.g.,a cancer specimen). Non-limiting examples of these mutations includenonsense mutations, missense mutations, frameshift mutations, or splicesite mutations that lead to gene variants that code for non-functionalor instable protein variants, or proteins with changed structure orfunction. Specific examples of these naturally occurring mutations aredepicted in Table I, such as the 3051delA, 2139+2T→C or 3055A→Tmutations. Other examples of these mutations are described in Table 4,such as 1949T→C or 2647A→G mutations. These mutations can be eithersomatic or germline mutations. In many cases, an EPHB2 gene comprisingone such mutation encodes an impaired EphB2 protein. As used herein, animpaired EphB2 protein has a reduced activity or a total loss ofactivity as compared to a wild type EphB2 protein. An impaired EphB2protein can also be less stable than the wild type protein such that thelevel of activity of the impaired EphB2 protein in cancer cells issubstantially lower than that of the wild type protein in nonmalignantcontrol cells.

Any method known in the art can be used to detect mutations in the EPHB2gene. Each mutation can be detected at the genomic sequence level, theRNA transcript level, or the polypeptide level. Methods suitable forthis purpose include, but are not limited to, conventional nucleicacid/polypeptide sequencing, fluorescent in situ hybridization (FISH),pulsed field gel electrophoresis (PFGE) analysis, high performanceliquid chromatography (HPLC), mass spectrometry, differentialhybridization based platforms, Southern blot analysis, gel mobilityassays such as single strand conformation polymorphisms (SSCP),restriction fragment length polymorphism or RFLP, RNase protectionassay, allele-specific oligonucleotide (ASO), protein truncation test(PTT) or immunoassays using antibodies specific for the mutated EphB2proteins but not the wild type proteins. Functional assays for EphB2proteins can also be used to evaluate mutations in the EPHB2 gene.

In another embodiment, the prognosis of a cancer in a patient ofinterest is assessed by detecting the presence or absence ofinactivation or deletion of one or more alleles of the EPHB2 gene in abiological sample of the patient (e.g., a cancer specimen). Methodssuitable for this purpose are well known in the art. Gene inactivationcan occur at the genomic level, the transcriptional level, or thetranslational level. Exemplary mechanisms of gene inactivation include,but are not limited to, genomic deletions, genomic mutations, aberrantmethylation, or epigenetic silencing. The inactivating mutations canoccur, for example, in the 5′ or 3′ regulatory sequences, the exons, orthe introns of the EPHB2 gene. These mutations can suppress thetranscription, translation, or protein function of the EPHB2 gene, ordestabilize the RNA transcript or protein product of the gene. Othergene inactivation mechanisms are also contemplated by the presentinvention.

In still another embodiment, the prognosis of a cancer in a patient ofinterest is evaluated by detecting the presence or absence of a reducedlevel of expression of the EPHB2 gene, or a reduced level of activity ofthe EphB2 protein, in a biological sample of the patient (e.g., a cancerspecimen). In many cases, the level of expression of the EPHB2 gene (orthe level of activity of the EphB2 protein) in the patient of interestis measured relative to the expression level of the wild type EPHB2 gene(or the activity level of the wild type EphB2 protein) in disease-freesamples or nonmalignant control cells. These disease-free samples ornonmalignant control cells are prepared from disease-free subjects usingthe same type of tissue as the biological sample of the patient ofinterest. Any method known in the art can be used to determine the levelof expression of the EPHB2 gene, or the level of activity of the EphB2protein. In one example, the expression level of the EPHB2 gene isdetermined by measuring the level of the RNA transcripts of the gene.Methods suitable for this purpose include, but are not limited to,quantitative RT-PCR, Northern Blot, in situ hybridization,slot-blotting, nuclease protection assays, or nucleic acid arrays. Inanother example, the expression level of the EPHB2 gene is determined bymeasuring the EphB2 protein level. Non-limiting examples of methodssuitable for this purpose include immunoassays (such as ELISA, RIA,FACS, or Western Blot), 2-dimensional gel electrophoresis, massspectrometry, or protein arrays.

Progressive changes in the EPHB2 gene are often associated with theprogression of the cancer. Therefore, the existence of abnormalities ontwo alleles of the EPHB2 gene is frequently indicative of a moreadvanced stage or a poorer prognosis of the cancer as compared to theexistence of abnormalities on only one allele of the gene. Likewise, theexistence of abnormalities on one allele of the EPHB2 gene is suggestiveof a more advanced disease stage or a poorer prognosis of the cancer ascompared to the absence of abnormalities in the EPHB2 gene.

In one example, the prognosis of a cancer in a patient of interest ispredicted by detecting the presence or absence of bi-allelicinactivation/deletion of the EPHB2 gene in a biological sample of thepatient (e.g., a cancer specimen). In another example, the cancer isprognosticated by detecting the presence or absence of a mutation on oneallele of the EPHB2 gene (e.g., a mutation selected from Table I orTable 4) and inactivation or deletion of the other allele of the EPHB2gene.

The above-described abnormalities can also be used for predicting acancer patient's response to a therapeutic treatment, or for staging thecancer in the patient. The presence of one such abnormality in abiological sample of the patient (e.g., a primary or metastatic cancerspecimen or a non-cancerous tissue sample) is indicative of a poorresponse of the cancer patient to the therapeutic treatment, and theabsence of such an abnormality is indicative of a good response of thecancer patient to the therapeutic treatment. Likewise, the presence ofone such abnormality in a biological sample of the patient is indicativeof an advanced stage of the cancer, and the absence of suchabnormalities suggests that the cancer in the patient is at an earlystage. The ability to accurately stage a cancer allows one to selectappropriate treatments for the cancer patient. Any type of biologicalsample that is suitable for cancer prognosis can also be used for cancerstaging or predicting cancer patient response to therapeutic treatments.

The present invention also features methods for diagnosis of cancer orcancer predisposition in a patient of interest. The methods comprisedetecting the presence or absence of an abnormality in a biologicalsample of the patient of interest, where the presence of the abnormalityis indicative of a cancer, or a predisposition thereto, in the patientof interest. Any abnormality or biological sample described above can beused for cancer diagnosis. In one embodiment, the abnormality beingdetected is a mutation selected from Table 1 or Table 4, such as the3055A→T (or K1019X) mutation, and the biological sample is a bloodsample of the patient of interest.

In addition, the present invention features methods for monitoring orevaluating the effectiveness of a treatment of a cancer in a patient ofinterest. These methods comprise monitoring an abnormality in thepatient of interest during the course of the treatment of the patient,where elimination or alleviation of the abnormality in the patient ofinterest during the course of the treatment is indicative of theeffectiveness of the treatment for the patient of interest. Anyabnormality or biological sample described above can be used to assessor monitor the effectiveness of a cancer treatment.

In another aspect, the present invention provides methods fordiagnosing, prognosticating, or staging a cancer in a patient ofinterest. These methods comprise detecting the presence or absence of adysfunctional EPHB2 gene in a biological sample of the patient ofinterest, where the presence of the dysfunctional EPHB2 gene in thebiological sample is indicative of the presence, a predisposition, apoor prognosis, or an advanced stage of the cancer in the patient ofinterest. As described above, a suitable biological sample can includeprimary or metastatic cancer cells. It can also be a sample preparedfrom non-cancerous tissues. In many embodiments, the dysfunctional geneencodes an impaired EphB2 protein, or has a reduced or abolished levelof expression. In one example, the dysfunctional gene includes amutation selected from Table 1 or Table 4. In another example, bothalleles of the EPHB2 gene are dysfunctional (e.g., inactivated, deleted,or encoding impaired EphB2 proteins).

In another aspect, the present invention provides methods foridentifying or screening for drug candidates that are capable ofreversing or alleviating a cellular abnormality caused by adysfunctional EPHB2 gene or an impaired or imbalanced EphB2-mediatedsignal transduction pathway. These methods comprise the steps of:contacting a candidate molecule with a cell, the cell comprising thedysfunctional EPHB2 gene or the impaired or imbalanced EphB2-mediatedsignal transduction pathway which causes an abnormal growth or survivalof the cell; and evaluating the growth or survival of the cell in thepresence of the candidate molecule. A suppression of the abnormalsurvival or growth of the cell in the presence of the candidatemolecule, as compared to in the absence of the candidate molecule,indicates that the candidate molecule is capable of correcting oralleviating the abnormal survival or growth of the cell caused by thedysfunctional EPHB2 gene.

In many embodiments, a candidate molecule thus identified is capable ofsuppressing the abnormal survival or growth of the cell under aspecified condition, but does not suppress the growth of nonmalignantcontrol cells under the same condition. The nonmalignant control cellspreferably are isolated from the same type of tissue as the cell beinginvestigated.

In one embodiment, the cell that comprises the dysfunctional EPHB2 geneor the impaired EphB2-mediated pathway is a prostate cancer cell, abreast cancer cell, a colon cancer cell, or a neuroblastoma cell. Thedysfunctional EPHB2 gene includes a mutation selected from Table 1 orTable 4, or encodes an impaired EphB2 protein, or has a reduced orabolished level of expression. In some cases, the effect of the impairedor imbalanced EphB2-mediated signal transduction pathway is similar tothat of a loss-of-function EPHB2 gene, and the impairment or imbalanceof the pathway can be repaired or improved by introducing a wild typeEPHB2 gene.

Drug candidates capable of correcting or alleviating a cellularabnormality caused by a dysfunctional EPHB2 gene can also be identifiedaccording to the following method: introducing an expression vector intoa cell, the cell comprising the dysfunctional EPHB2 gene which causes anabnormal survival or growth of the cell, and the expression vectorencoding a polypeptide or polynucleotide of interest; expressing theexpression vector in the cell to produce the polypeptide orpolynucleotide; and evaluating the survival growth of said cell in thepresence of the expressed polypeptide or polynucleotide. A suppressionof the abnormal survival or growth of the cell in the presence of theexpressed polypeptide or polynucleotide, as compared to in the absenceof the expressed polynucleotide or polypeptide, indicates that thepolypeptide or polynucleotide is capable of reversing or alleviating theabnormal survival or growth caused by the dysfunctional EPHB2 gene.

In still another aspect, the present invention provides methods foridentifying mutations in genes which, when combined with a dysfunctionalEPHB2 gene, suppress abnormal cell survival or growth. These methodscomprise: providing a plurality of cells, each of which comprises thedysfunctional EPHB2 gene and a mutation in a corresponding gene; andevaluating the survival or growth of each said cell. A reduced survivalor growth rate in one of these cells, as compared to that of a controlcell, which comprises the dysfunctional EPHB2 gene but not the mutationin the corresponding gene, indicates that the mutation in thecorresponding gene, when combined with the dysfunctional EPHB2 gene, cansuppress the survival or growth of the cell.

Similarly, the present invention features methods for identifying geneshaving synthetic lethal interactions with EPHB2 gene alterations. Thesemethods comprise: suppressing the expression of a gene of interest in acell that comprises a dysfunctional EPHB2 gene; and evaluating thesurvival or growth of the cell upon suppression of the gene of interest.A reduced survival or growth rate of the cell, as compared to that of acontrol cell which comprises the dysfunctional EPHB2 gene but in whichthe gene of interest is not suppressed, indicates that the gene ofinterest has a synthetic lethal interaction with the dysfunctional EPHB2gene.

Any dysfunctional EPHB2 gene described herein can be used for screeningfor synthetic lethal partners. The suppression of a gene of interest canbe achieved by using chemical compounds, antisense RNAs, or RNAisequences.

The present invention also features methods for identifying or screeningfor physical, biological, or chemical agents capable of inhibiting aprotein activity of a gene that has a synthetic lethal interaction withEPHB2 alterations. The methods comprise: contacting a candidate moleculewith a protein product of the synthetic lethal gene; detecting anactivity of the protein product in the presence or absence of saidagent. A reduced level of the activity of the protein product in thepresence of the candidate molecule, as compared to in the absence of thecandidate molecule, is indicative of the capability of the candidatemolecule to inhibit the synthetic lethal gene.

In yet another aspect, the present invention features methods forinhibiting an abnormal growth or survival of a cell caused by adysfunctional EPHB2 gene. The methods comprise introducing a wild typeEPHB2 protein, or an expression vector encoding the same, into the cell.

In addition, the present invention features pharmaceutical compositionscomprising a therapeutically or prophylactically effective amount of awild type EphB2 protein, or an expression vector encoding the same. Apharmaceutical composition of the present invention can also include apharmaceutically acceptable carrier.

The present invention also features antibodies specifically recognizingan EphB2 protein with a mutation selected from Table 1 or Table 4, butnot wild type EphB2 proteins. Prognostic or diagnostic kits comprisingan antibody of the present invention are also provided. In addition, thepresent invention features immunotoxin or cytotoxic cells specificallyrecognizing mutated EphB2 proteins but not the wild type proteins.

Moreover, the present invention features polynucleotides capable ofhybridizing under stringent conditions to an RNA transcript, or thecomplement thereof, of an EPHB2 gene with a mutation selected from Table1 or Table 4, but not RNA transcripts, or the complements thereof, ofwild type EPHB2 genes. The present invention also features prognostic ordiagnostic kits comprising a polynucleotide of the present invention.

Furthermore, the present invention features methods for identifyingcandidate tumor suppressor genes. These methods comprise: detecting geneexpression changes in cancer cells after inhibition of the nonsensemediated RNA decay pathway in the cancer cells; detecting geneexpression changes in nonmalignant control cells after inhibition of thenonsense-mediated RNA decay pathway in these cells; comparing the geneexpression changes in the cancer cells with the changes in thenonmalignant control cells to identify genes whose expression levels areincreased in the cancer cells but not in the nonmalignant control cellsafter inhibition of the nonsense-mediated RNA decay pathway; detectinggenomic regions that are deleted in the cancer cells but not in diseasefree cells; and selecting from the identified genes a gene whose locusmaps within one of the deleted genomic regions. A gene thus selected isa candidate tumor suppressor gene.

In one embodiment, the nonsense-mediated RNA decay pathway is inhibitedin the cancer cells (e.g., by emetine), followed by suppression of newRNA synthesis (e.g., by actinomycin D) to distinguishpost-transcriptional shifts in mRNA stability. The levels of RNAtranscripts in these cancer cells can then be measured by using anyconventional means including cDNA or oligonucleotide microarrays. As acontrol, new RNA synthesis, but not the nonsense mediated RNA decaypathway, is inhibited in the same type of cancer cells. The levels ofRNA transcripts in these control cancer cells are similarly measured andcompared to the levels of RNA transcripts in the cancer cells (in whichboth nonsense-mediated RNA decay and RNA synthesis are blocked) todistinguish gene expression changes caused by the suppression of thenonsense-mediated RNA decay pathway.

The gene expression changes in the nonmalignant control cells can besimilarly measured and compared to the gene expression changes in thecancer cells to identify mutation induced transcript stabilizationevents, as opposed to drug-induced gene expression changes (e.g.,induced by emetine or actinomycin D).

In another embodiment, comparative genomic hybridization on cDNAmicroarrays is employed to detect genomic regions that are deleted incancer cells but not in disease-free cells. These deleted genomicregions can be compared to the genes whose expression levels arestabilized by inhibition of the nonsense-mediated RNA decay pathway.Among these genes, potential tumor suppressor genes are identified asthose whose loci map within the deleted genomic regions.

The present invention further features methods for assessing cancer riskin a subject of interest. The methods comprise detecting the presence orabsence of an abnormality in a biological sample of the subject ofinterest, where the presence of the abnormality is indicative of anincreased risk of a cancer in the subject of interest, as compared tohealthy subjects or subjects without such an abnormality. Non-limitingexamples of abnormalities suitable for this purpose include: a mutationin EPHB2, where the mutated EPHB2 encodes an impaired EphB2 protein;inactivation of at least one allele of EPHB2; deletion of at least oneallele of EPHB2; a reduced level of expression of EPHB2; a reduced levelof activity of an EphB2 protein; or any combination thereof.

In one embodiment, the cancer being assessed is prostate cancer, and theabnormality is a mutation selected from Table 4. The mutation can be asomatic or germline mutation. In a specific example, the abnormality isa 3055A→T mutation on one or two alleles of the EPHB2 gene. In anotherspecific example, the subject of interest is an African American.

Any type of biological samples can be used for evaluating the risk ofcancer in a subject of interest. In one example, the biological sampleis a blood sample or another available source of germline DNA.

Other features, objects, and advantages of the present invention areapparent in the detailed description that follows. It should beunderstood, however, that the detailed description, while indicatingembodiments of the present invention, is given by way of illustrationonly, not limitation. Various changes and modifications within the scopeof the invention will become apparent to those skilled in the art fromthe detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings are provided for illustration, not limitation.

FIG. 1 illustrates relative changes in transcript levels resulting fromEmetine mediated NMD blockade. DU 145 and PC-3 cell lines werepretreated with Emetine to block the NMD pathway and then treated withActinomycin D to block new transcription. Transcripts were detected andmeasured by cDNA microarray (a, c, and e) and validated by quantitativeRTPCR analysis (b, d, and f).

FIG. 2 shows integration of microarray data from CGH and NMD blockadeanalysis for genome-wide prioritization of the candidate TSGs. Thecumulative base pair location for the entire human genome beginning with1 pter (x axis) was used to map each gene and its copy number statusfrom the CGH microarray analysis of DU 145 cells (line on lower half),as well as the corresponding NMD microarray data for genes with anincreased NMD ratio in DU 145 cells (points on upper half).

FIG. 3 demonstrates the localization of observed mutations on the EphB2protein. The key physical structures and domains of EphB2 areillustrated, and the locations of exemplary mutations found in prostatecancer are also indicated. Two of the mutations occur in theextracellular part of the protein and six in the intracellular part. Thekinase domain, which is critical for receptor signaling, appears to bethe most frequently targeted site. The frameshift mutation 3051delAextends the protein by 72 amino acids, as indicated.

FIG. 4 illustrates the relative level of expression of EPHB2, ascompared to the β-actin gene, in a number of cancer lines. Total RNA wasextracted using Qiagen RNeasy Mini Kit Spin Columns. cDNA wassynthesized using Invitrogen's two-step Thermoscript RT-PCR system. They-axis indicates the relative expression level of EPHB2, and the x-axislists each cell line being investigated.

FIG. 5 shows the results of quantitative RT-PCR analysis of theexpression level of EPHB2 in different cancer cells. The analysis wascarried out using ABI's AoD TaqMan Assays (EphB2: Hs00363096_ml,β-actin: Hs99999903_ml) and ABI's Universal PCR Master Mix (catalognumber 434437).

FIG. 6A indicates that EphB2 expression suppresses the growth of DU 145prostate cancer cells. DU 145 cells, with a single truncated form ofEphB2, were transfected (from left to right) with control vector, twohuman wild type EphB2 expression constructs (EphB2/A and EphB2/B,representing independent subclones), murine wild type EphB2 (mEphB2) orwild type p53 expression constructs (P53). Quantification of cell growthwas measured with Cell Titer Blue and presented as a percentage growthof cells transfected with control vector alone. Expression of EphB2protein was verified by western blotting (inset).

FIG. 6B shows that all EphB2 clones described in FIG. 4A reduced thegrowth of DU 145 cells and the number of colonies formed as efficientlyas did an expression construct of p53 (which carries the missensemutations P223L and V274F in DU 145).

DETAILED DESCRIPTION

The present invention combines nonsense-mediated RNA decay microarraysand array-based comparative genomic hybridization (CGH) for genome-wideidentification of genes with bi-allelic inactivation involving nonsensemutations and loss of the wild type allele. This approach resulted inthe discovery of mutations in the receptor tyrosine kinase gene EPHB2.The DU 145 prostate cancer cell line, originating from a brainmetastasis, carries a truncating mutation of the EPHB2 gene and adeletion of the remaining allele. Additional frameshift, splice site,missense and nonsense mutations are present in clinical prostate cancersamples. Transfection of DU 145 cells, which lack functional EphB2, withwild type EPHB2 suppresses clonogenic growth. Taken together withstudies implicating a critical role for EphB2 in cell migration andmaintenance of normal tissue architecture, the findings of the presentinvention indicate that EPHB2 has tumor-suppression activity and thatmutational inactivation or impairment of EPHB2 plays an important rolein cancer progression and metastasis.

In one aspect, the present invention provides methods for prognosis,diagnosis, or assessment of the progression of a cancer in a patient ofinterest. These methods include detecting the presence or absence of analteration in the EPHB2 gene or an abnormality in an EphB2-associatedsignal transduction pathway. An inactivating or impairing mutation inthe EPHB2 gene or an abnormality in its associated pathway is indicativeof the prognosis, diagnosis, or progression stage of the cancer in thepatient of interest.

In another aspect, the present invention provides pharmaceuticalcompositions for treating or preventing cancers that are characterizedby a dysfunctional or impaired EPHB2 gene or a deregulatedEphB2-mediated signal transduction pathway. Non-limiting examples ofcancers that are amenable to the present invention include prostatecancer, breast cancer, colon cancer, and neuroblastoma. In oneembodiment, a pharmaceutical composition of the present inventioncomprises a therapeutically or prophylactically effective amount of awild type EphB2 protein, or a biologically active fragment, variant ormimic thereof. In another embodiment, a pharmaceutical composition ofthe present invention comprises a therapeutically or prophylacticallyeffective amount of a nucleic acid molecule encoding a wild type EphB2protein or a biologically active fragment or variant thereof.

Various aspects of the invention are described in further detail in thefollowing subsections. The use of subsections is not meant to limit theinvention. Each subsection may apply to any aspect of the invention. Inthis application, the use of “or” means “and/or” unless statedotherwise.

I. IDENTIFICATION OF EPHB2 AS A TUMOR-SUPPRESSOR GENE

The present invention combined results from NMD microarray experimentshighlighting putative nonsense mutations with high-resolution data ondeleted genomic regions in cancer cell lines obtained with array-basedcomparative genomic hybridization. This integrated approach, whichfocused on bi-allelic gene inactivation events, was employed to identifycandidate TSGs in prostate cancer. DU 145, PC-3 and LNCaP prostatecancer cell lines were pretreated with Emetine (which inhibits the NMDpathway) and, then subjected to Actinomycin D to block new mRNAsynthesis and to distinguish post-transcriptional shifts in mRNAstability, which indicate the presence of a nonsense mutation cDNAmicroarrays were used to measure changes in transcript levels inEmetine-treated cells as compared to untreated cells. Correspondinganalyses were also carried out with non-malignant control cells in orderto distinguish drug-induced gene expression changes frommutation-induced transcript stabilization events. Known nonsensemutations including the C39X mutation in the MLHI gene in the DU 145cell line and the A138X mutation in the TP53 gene in the PC-3 cell linewere used as positive controls for the optimization of technology andvalidation of the strategy.

As predicted, the normalized NMD microarray ratios for both MLH1 andTP53 were elevated in the specific cell lines harboring these truncatingmutations, but not in the cell lines without such mutations. Theaccumulation of these mutated transcripts was validated by quantitativeRT-PCR (FIG. 1). Both the microarray and quantitative RT-PCR results inFIG. 1 represent relative expression ratios (Emetine treated versusuntreated), which were then averaged across the time course. The plotsshow microarray data for MLHI (a), p53 (c), and EphB2 (e) for DU 145 andPC-3 cell lines. Similarly, quantitative RT-PCR validation using genespecific primers is shown for MLHI (b), p53 (d), and EphB2 (f) for thesame cell lines. Increased relative transcript levels were only observedin cell lines with corresponding known (MLHI in DU 145 and p53 in PC-3)or new (EphB2 in DU 145) nonsense mutation. The differences betweenemetine-treated and untreated samples for all mutations, in bothmicroarray and quantitative RT-PCR experiments, were statisticallysignificant (p<0.01) as measured by both parametric t-test (two-tailed)and non-parametric Wilcoxon Rank Sum Test. In addition, cDNAmicroarray-based CGH indicated that the TP53 locus was deleted in thePC-3 cells, and MLHI was deleted in the DU 145 cancer cells.

Given the validation of the approach, the same strategy was applied toidentify new candidate genes in three prostate cancer cell lines (DU145, PC-3 and LNCaP). 0.4% of the genes on the array (45 in DU 145,65 inPC-3, and 68 for LNCaP) showed an overall increase in normalized NMDratio of at least three-fold in these cell lines. These genes wereadditionally prioritized by selecting candidates that were located indeleted regions of the genome in the corresponding cell line. Thisresulted in 36 prioritized candidate genes (9 for DU 145, 10 for PC-3and 17 for LNCaP) for analysis by DNA sequencing to detect putativemutations in each cell line.

One of the candidates selected for mutation analysis is based on thesecriteria was the EPHB2 gene. Ephrin receptors make up the largest familyof receptor tyrosine kinases (RTKs) and can mediate bi-directionalsignaling through their membrane-associated ephrin (Eph) ligands. Basedon their structures and sequence relationships, ephrins are divided intothe ephrin-A (EFNA) class, which are anchored to the membrane by aglycosylphosphatidylinositol linkage, and the ephrin-B (EFNB) class,which are transmembrane proteins. Eph receptors are also divided into 2groups based on the similarity of their extracellular domains and theiraffinities for binding to ephrin-A or ephrin-B ligands.

EPHB2 gene is also known as DRT, ERK, HekS, EPHT3 or Tyro5 and encodes areceptor for ephrin-B family members. At least two alternatively splicedEphB2 isoforms have been reported. EphB2 isoform 1 precursor (variant 1)encodes a longer isoform and comprises the amino acid sequence depictedin SEQ ID NO: 1 (Entrez accession number NP_(—)059145). Variant 1includes a signal peptide (amino acid residues 1-18), an Ephrin receptorligand binding domain (amino acid residues 20-197), two fibronectin typeIII domains (amino acid residues 332-419 and 439-520, respectively), atyrosine kinase catalytic domain (amino acid residues 615883), and asterile alpha motif or SAM (amino acid residues 910-977). An exemplarycDNA sequence that encodes variant 1 is depicted in SEQ ID NO: 2 (Entrezaccession number NM_(—)017449), within which the sequence fromnucleotide 19 to nucleotide 3,183 is the codon sequence.

EphB2 isoform 2 precursor (variant 2) includes an alternate in-framesegment, resulting in a shorter protein that has a distinct C-terminus,compared to variant 1. The amino acid sequence of variant 2 isillustrated in SEQ ID NO: 3 (Entrez accession number NP_(—)004433). Thedomain structure of variant 2 is similar to that of variant 1. Anexemplary cDNA sequence that encodes variant 2 is depicted in SEQ ID NO:4 (Entrez accession number NM_(—)004442), within which the sequence fromnucleotide 19 to nucleotide 2,979 is the codon sequence.

Eph receptors regulate intracellular signaling pathways involved in cellgrowth, migration, adhesion, and polarity. Eph receptors and ephrins arefrequently expressed in complementary patterns that correlate withcellular boundaries during embryonal development. Eph-ephrin signalingcan prevent the inappropriate intermingling of distinct cells inculture, and is involved in vascular modeling, axon guidance andepithelial-mesenchymal transitions. In addition, EphB2 and EphB3 controlthe correct positioning of cells in both the embryonic intestinalepithelium and the intestinal crypts. Disruption of the murineEPHB2/EPHB3 genes has been shown to interfere with normal cellularorganization in the crypts, resulting in a loss of normal cellpositioning and aberrant mixing of different cell types. Eph receptorscan also inhibit Ras-MAP kinase signaling and their inactivation maycontribute to the mitogenic activity of this pathway, a featuresupported by the observation that wild type EphB2 can suppressclonogenic growth.

EPHB2 gene showed an increased normalized NMD microarray ratio in the DU145 cells, but not in PC-3 cells (FIG. 1). This suggestspost-transcriptional stabilization of mutated EphB2 mRNA following NMDblockade in the DU 145 cells. This differential expression was confirmedby quantitative RT-PCR (FIG. 1). Additionally, EPHB2 mapped to a deletedregion at 1p36 by CGH microarray data further suggesting bi-allelicinactivation of the gene (FIG. 2). In FIG. 2, the CGH plot was generatedusing a moving average of the mean ratios of 30 consecutive clones. Thedeleted regions are indicated with horizontal dark grey bars. Genes withpositive mean normalized NMD-ratio above 3 fold are mapped and plottedto their corresponding location. Genes with a positive NMD-ratio mappingwithin deleted loci such as MLH1 and EPHB2 (circled) were prioritized.Vertical grey bars indicate the deleted regions corresponding to theEPHB2 and MLHI loci. Similar results were obtained for PC-3 in which atruncating mutation in TP53 was associated with both a positive NMDmicroarray ratio and mapped to a deleted region of 17p in PC-3.

Notably, the deleted chromosomal locus at 1p36 has been linked tohereditary prostate cancer. See Gibbs, et al, AM J HUM GENET, 64:776-787 (1999). Sequencing confirmed that the DU 145 cell line had ahemizygous nonsense mutation Q723X (Table 1) that truncates EphB2 at thekinase domain (FIG. 3) and is predicted to lead to a complete loss ofreceptor signaling.

To determine the relevance of this finding in clinical disease, DNAspecimens from uncultured, clinical prostate tumors (including 33primary and 62 metastasis specimens) were screened for EPHB2 mutations.Several mutations were confirmed (Table 1). Most notable was aframeshift mutation 3051 delA that was found in three independentmetastatic lesions (subdural, humerus, sternum) from the same patient.The mutation was not present in the normal liver sample of the sameindividual, suggesting that this represented a somatic mutation. Thismutation alters the reading frame at the end of isoform 1 of EPHB2,resulting in an extension of the protein by 72 amino acids. Also ofinterest was a non-coding mutation found in a prostate cancer bonemetastasis. This splice site mutation (2139+2T→C) destroys the consensussplice donor (GT) of exon 11, which is 100% conserved in eukaryotes. Inaddition, four missense mutations were observed (Table 1 and FIG. 3).The nucleotide numbering in Table 1 is with reference to the firstnucleotide of the start codon (i.e., nucleotide 19 in SEQ ID NOs: 2 or4).

TABLE 1 Mutation in EPHB2 Loss of Nucleotide Amino Acid wild type NumberSample change change Effect allele of cases origin Frequency^(a) 596G→AR199H Missense No 1 Primary 0/111 835G→T A279S Missense Yes 1 Metastasis0/183 2035G→A D679N Missense No 2 Primaries 0/246 2139 + 2 T>C — Splicesite Yes 1 Metastasis 0/150 2167C→T Q723X Nonsense Yes 1 DU 145 0/1002726C→T T909M Missense No 1 Primary N/A 3055A→T^(b) K1019X^(b) NonsenseNo 1 Metastasis 4/231 3051delA^(b) — Frameshift No 1 Metastasis 0/231^(a)Frequency in normal population ^(b)Isoform 1

Deregulation of the EPHB2 gene was also observed in other types ofcancers. FIG. 4 illustrates the relative level of expression of EPHB2 indifferent cancer cell lines, as compared to the β-actin gene. SKBR-3, ahuman breast cancer cell line, exhibits the lowest level of expressionof EPHB2 among the cell lines being investigated. MCF-7, another humanbreast cancer cell line, also shows a significantly lower level ofexpression of EPHB2 as compared to PC-3 cells. Quantitative RT-PCRanalysis, as depicted in FIG. 5, also demonstrates that SKBR-3 cellshave the lowest level of expression of EPHB2 among the cell lines beingtested.

The above data demonstrates that EphB2 has tumor-suppression activity,and the deregulation or inactivation of the gene is associated with avariety of cancers (e.g., prostrate or breast cancers). To furthersupport the EphB2 tumor-suppression activity, wild type EphB2 constructswere transfected into the DU 145 cell line (which does not havefunctional EphB2) and the effects on cell growth using colony formationassays were measured. Two human and one murine wild type EphB2expression constructs were all able to suppress the growth and colonyformation of DU 145 cells. The suppression was as efficient as that seenwith wild type p53 transfection, a TSG that is inactivated by twomissense mutations in this cell line (FIG. 4). These findings furthersupport the functional significance of EphB2 mutations in prostatecancer progression.

A variety of genetic alterations have been observed throughout theunstable genomes of cancer cells, and these tend to accumulate inadvanced stages of disease. However, recurrent sequence mutations in thesame gene observed in multiple clinical samples are rare. Multiplerecurrent mutations, especially if they are inactivating in nature andassociated with loss of heterozygosity (LOH), indicatedisease-associated changes that accumulate because they confer aselective growth advantage to neoplastic cells. The genetic andfunctional data presented herein provide compelling evidence of apathogenic role of mutations in EphB2 in prostate and breast cancers.

The integrated strategy of the present invention for the genome-widescreening of inactivating mutations in cancer led to the discovery ofdeleterious mutations of EPHB2 in a prostate cancer cell line.Investigation of clinical samples validated the mutational inactivationof EPHB2 in about 8% of primary and metastatic prostate cancers. Theexpression of wild type EphB2 significantly suppresses the growth of theDU 145 cell line. Mutations in the EPHB2 gene were also associated withbreast cancer and neuroblastoma. These observations support EphB2′ srole as a TSG involved in cancer progression. Loss of EphB2 signalingthrough mutational inactivation may impact on multiple phenotypicaspects of cancer, such as aberrant growth, invasion and metastasis.

II. DIAGNOSIS, PROGNOSIS, STAGING, AND ASSESSMENT OF RISK ORPREDISPOSITION OF CANCERS

The EPHB2 gene or its expression products can be used as biologicallymarkers for the diagnosis, prognosis, staging, or assessment of risk orpredisposition of cancer. The present invention identifies the EPHB2gene as a tumor-suppressor gene. Recurrent sequence mutations in thegene have been observed in a variety of cancers including, but notlimited to, prostate cancer, breast cancer, and neuroblastoma.Therefore, an alteration in EPHB2 is indicative of the risk,predisposition, or disease status of cancer in a subject of interest.

In one example, a subject of interest has inherited a germline EPHB2mutation (e.g., a mutation depicted in Table 1) and, therefore, is proneto develop cancer. The early detection of such a mutation allowspreventive treatment of the subject to avert or delay the development ofcancer. In another example, a subject of interest has EPHB2 mutation(s)in one or two alleles. Somatic EPHB2 mutation(s) in only one allele isoften indicative of an early neoplastic state, while somatic EPHB2mutations in both alleles are frequently suggestive of a late neoplasticstate. Accordingly, different prevention or treatment approaches can beselected for a patient of interest based on the extent or severity ofEPHB2 mutations. In a further example, alterations in EPHB2 are used incombination with other clinical evidence for the diagnosis or assessmentof the risk of cancer.

The present invention also features prognostic use of the EPHB2 gene.For instance, a specified mutation in the EPHB2 gene may be correlatedwith good or poor prognosis of cancer patients. Consequently, thepresence or absence of the specified EPHB2 mutation in a patient ofinterest is indicative of the prognosis or clinical outcome of thepatient. Methods suitable for correlating EPHB2 gene alterations withpatients' prognoses include, but are not limited to, thenearest-neighbor analysis, the significance method of microarrays, orother supervised or unsupervised learning or clustering algorithms. Theability to prognosticate a patient of interest allows one to select oroptimize favorable treatments for the patient.

Any method known in the art can be used to detect alterations orabnormalities in the EPHB2 gene. As used herein, an alteration orabnormality in the EPHB2 gene includes any structural or functionalchange in the gene that results in a change in the expression orfunction of the encoded protein as compared to a wild type EphB2protein. Exemplary wild type EphB2 proteins include those depicted inSEQ ID NOs: 1 and 3. In many cases, the alterations or abnormalities inEPHB2 include, but are not limited to, deletions, insertions,duplications, or point mutations in the coding or noncoding regions ofthe EPHB2 gene. Deletions may be of the entire gene or only a portion ofthe gene. Point mutations may result in stop codons, frameshiftmutations, or amino acid substitutions. Alterations can occur in one orboth alleles of the EPHB2 gene, and each affected allele may include oneor more alterations. Each EPHB2 allele may have the same or differentalterations.

An alteration in the EPHB2 gene may result in a dysfunctional orinactivated EphB2 protein. The alteration may also lead to thereduction, abolishment, dysfunction, or deregulation of the expression(e.g., transcription or translation) of the gene. For instance, pointmutational events can occur in regulatory regions, such as in thepromoter of the gene, leading to loss or diminution of expression of themRNA. Point mutations can also abolish proper RNA processing, leading toloss of expression of the EPHB2 gene product, or a decrease in mRNAstability or translation efficiency. The present invention, however,does not exclude the possibility that a tumor-causing mutation mayresult in an elevated level or activity of the EPHB2 gene or itsexpression product(s).

An alteration in EPHB2 can be a somatic or germline mutation. Somaticmutations are those that occur only in certain non-germline tissues andare not inherited in the germline. Germline mutations can be found inany body tissue and are inherited. In many cases, a cell takes a geneticstep toward oncogenic transformation when one allele of a tumorsuppressor gene is inactivated due to inheritance of a germline lesionor acquisition of a somatic mutation. The inactivation of the otherallele of the gene may involve a somatic micromutation or chromosomalallelic deletion that results in loss of heterozygosity (LOH). Incertain instances, both copies of a tumor-suppressor gene may be lost byhomozygous deletion.

Mutations or alterations in EPHB2 can be detected at various levels,such as the genome level, the transcriptome level, or the proteomelevel. Techniques that are suitable for detecting mutations oralterations in EPHB2 include, but are not limited to, fluorescent insitu hybridization (FISH), direct DNA or RNA sequencing (e.g.,denaturing high-performance liquid chromatography or DHPLC), pulsedfield gel electrophoresis (PFGE) analysis, Southern blot analysis, gelmobility assays (e.g., single stranded conformation analysis (SSCA) orsingle-strand conformational polymorphism (SSCP)), restrictionenzyme-based assays (e.g., restriction fragment length polymorphism orRFLP), RNase protection assay, allele-specific oligonucleotide (ASO),dot blot analysis, hybridization using nucleic acid modified with goldnanoparticles and PCR-SSCP, DNA or protein microchip technology, massspectrometry analysis, Northern blot analysis, two-dimensionalelectrophoresis analysis, and immunoassays (e.g., Western blot analysis,Enzyme-Linked Immunosorbent Assay (ELISA), or radioimmune assay (RIA)).Other methods known in the art can also be used to detect mutations oralterations in EPHB2.

Any cell or tissue sample can be used to detect EPHB2 gene alterations.In one embodiment, a sample employed in the present invention is derivedfrom a tissue selected from prostate, breast, skin, muscle, facia,brain, endometrium, lung, head and neck, pancreas, small intestine,blood, liver, testes, ovaries, colon, skin, stomach, esophagus, spleen,lymph node, bone marrow, kidney, placenta, or fetus. In anotherembodiment, a sample employed in the present invention is a fluidsample, such as peripheral blood, lymph fluid, ascites, serous fluid,pleural effusion, sputum, cerebrospinal fluid, amniotic fluid, lacrimalfluid, stool, or urine. A sample being tested can be cancerous ornon-cancerous. It can include cells from one or more tissues.

In many embodiments, EPHB2 gene alterations are determined by detectingDNA sequence variations. Direct DNA sequencing, either manual sequencingor automated fluorescent sequencing, can detect sequence variation;Other methods include, but are not limited to, single strandedconformation analysis (SSCA), denaturing gradient gel electrophoresis;RNase protection assays, allele-specific oligonucleotides (ASOs), theuse of proteins which recognize nucleotide mismatches (such as the E.coli mutS protein), and allele specific PCR. SSCA detects a band whichmigrates differentially because the sequence change causes a differencein single-strand, intramolecular base pairing. DGGE detects differencesin migration rates of mutant sequences compared to wild type sequences,using a denaturing gradient gel. RNase protection involves cleavage ofthe mutant polynucleotide into two or more smaller fragments. In anexemplary allele-specific oligonucleotide assay, an oligonucleotide isdesigned which detects a specific sequence, and the assay is performedby detecting the presence or absence of a hybridization signal. In themutS assay, the protein binds only to sequences that contain anucleotide mismatch in a heteroduplex between mutant and wild typesequences. In one format of allele-specific PCR, primers are used whichhybridize at their 3′ ends to a particular EPHB2 mutation. If theparticular EPHB2 mutation is not present, an amplification product isnot observed.

Mismatches, according to the present invention, are hybridized nucleicacid duplexes in which the two strands are not 100% complementary. Lackof total homology may be due to deletions, insertions, inversions orsubstitutions. Mismatch detection can be used to detect point mutationsin the gene or its mRNA product. While these techniques are lesssensitive than sequencing, they are simpler to perform on a large numberof samples. An example of a mismatch cleavage technique is the RNaseprotection method, as described above. In one format, the methodinvolves the use of a labeled riboprobe complementary to the human wildtype EPHB2 gene coding sequence. The riboprobe and either mRNA or DNAisolated from the tissue of interest are annealed (hybridized) togetherand subsequently digested with the enzyme RNase A, which is able todetect some mismatches in a duplex RNA structure. If a mismatch isdetected by RNase A, it cleaves at the site of the mismatch. Thus, whenthe annealed RNA preparation is separated on an electrophoretic gelmatrix, if a mismatch has been detected and cleaved by RNase A, an RNAproduct will be seen which is smaller than the full length duplex RNAfor the riboprobe and the mRNA or DNA. The riboprobe need not be thefull length of the EPHB2 mRNA or gene but can be a segment of either. Ifthe riboprobe comprises only a segment of the EPHB2 mRNA or gene, itwill be desirable to use a number of these probes to screen the wholemRNA sequence for mismatches.

In similar fashion, DNA probes can be used to detect mismatches, throughenzymatic or chemical cleavage. Alternatively, mismatches can bedetected by shifts in the electrophoretic mobility of mismatchedduplexes relative to matched duplexes.

Genomic sequences of the EPHB2 gene which have been amplified by use ofPCR may also be screened using allele-specific probes. These probes arenucleic acid oligomers, each of which contains a region of the EPHB2gene sequence harboring a known mutation. For example, one oligomer maybe about 30 nucleotides in length, corresponding to a portion of theEPHB2 gene sequence. By use of a battery of such allele-specific probes,PCR amplification products can be screened to identify the presence of apreviously identified mutation in the EPHB2 gene. Hybridization ofallele-specific probes with amplified EPHB2 sequences can be performed,for example, on a nylon filter. Hybridization to a particular probeunder high stringency hybridization conditions indicates the presence ofthe same mutation in the tumor tissue as in the allele-specific probe.In one example, the hybridization conditions are selected such that an 8base pair stretch of a first nucleic acid (a probe) can bind to a 100%perfectly complementary 8 base pair stretch of nucleic acid whilesimultaneously preventing binding of said first nucleic acid to anucleic acid which is not 100% complementary, i.e., binding will notoccur if there is a mismatch. Methods for designing allele-specificprobes or PCR primers are well known in the art. Table 2 describesexemplary high stringency conditions that can be used for designing theprobes/primers of the present invention. In Table 2, hybridization iscarried out under a specified hybridization condition for about 2 hours,followed by two 15-minute washes under the corresponding wash condition.

TABLE 2 High Stringency Conditions Stringency Poly-nucleotide HybridHybridization Temperature Wash Temp and Condition Hybrid Length (bp)¹and Buffer^(H) Buffer^(H) A DNA:DNA >50 65° C.; 1xSSC -or- 65° C.;0.3xSSC 42°; 1xSSC; 50% formamide B DNA:DNA >50 T_(B)*; 1xSSC T_(B)*;1xSSC C DNA:RNA >50 67° C.; 1xSSC -or- 67° C.; 0.3xSSC 45° C.; 1xSSC,50% formamide D DNA:RNA >50 T_(p)*; 1SSX T_(p)*; 1SSX E RNA:RNA >50 70°C.; 1xSSC -or- 70° C.; 0.3xSSC 50° C.; 1xSSC, 50% formamide FRNA:RNA >50 T_(F)*; 1xSSC T_(F)*; 1xSSC ¹The hybrid length is thatanticipated for the hybridized region(s) of the hybridizingpolynucleotides. When hybridizing a polynucleotide to a targetpolynucleotide of unknown sequence, the hybrid length is assumed to bethat of the hybridizing polynucleotide. When polynucleotides of knownsequence are hybridized, the hybrid length can be determined by aligningthe sequences of the polynucleotides and identifying the region orregions of optimalsequence complementarity. ^(H)SSPE (1xSSPE is 0.15MNaCl, 10 mM NaH₂P0₄, and 1.25 mM EDTA, pH 7.4) can be substituted forSSC (1xSSC is 0.15M NaCl and 15 mM sodium citrate) in the hybridizationand wash buffers. T_(B)*~T_(F)*: The hybridization temperature forhybrids anticipated to be less than 50 base pairs in length should be5-10° C. less than the melting temperature (T_(m)) of the hybrid, whereT_(m) is determined according to the following equations. For hybridsless than 18 base pairs in length, T_(m)(° C.) = 2(# of A + T bases) +4(# of G + C bases). For hybrids between 18 and 49 base pairs in length,Tm (° C.) = 8.15 + 16.6(log₁₀Na⁺) + 0.41(% G + C) − (600/N), where N isthe number of bases in the hybrid, and Na⁺ is the concentration ofsodium ions in the hybridization buffer (Na⁺ for 1xSSC = 0.165M).

In one embodiment, the allele-specific probes or primers are designed tospecifically recognize an EPHB2 mutation described in Table 1 but notthe corresponding wild type sequence. The probes/primers can have anydesirable length. The probes/primers can be DNA, RNA, PNA, or a modifiedform thereof. The nucleotide residues in each probe can be eithernaturally occurring residues (such as deoxyadenylate, deoxycytidylate,deoxyguanylate, deoxythymidylate, adenylate, cytidylate, guanylate, anduridylate), or synthetically produced analogs that are capable offorming desired base-pair relationships. Examples of these analogsinclude, but are not limited to, aza and deaza pyrimidine analogs, azaand deaza purine analogs, and other heterocyclic base analogs, whereinone or more of the carbon and nitrogen atoms of the purine andpyrimidine rings are substituted by heteroatoms, such as oxygen, sulfur,selenium, and phosphorus. Similarly, the polynucleotide backbones of theprobes can be either naturally occurring (such as through 5′ to 3′linkage), or modified. For instance, the nucleotide units can beconnected via non-typical linkage, such as 5′ to 2′ linkage, so long asthe linkage does not interfere with hybridization. For another instance,peptide nucleic acids, in which the constitute bases are joined bypeptide bonds rather than phosphodiester linkages, can be used.

Changes in the genomic sequence of EPHB2 gene can also be detected usingSouthern hybridization, especially if the changes are grossrearrangements, such as deletions and insertions. A rapid preliminaryanalysis to detect polymorphisms in DNA sequences can be performed bylooking at a series of Southern blots of DNA cut with one or morerestriction enzymes, preferably a large number of restriction enzymes.Each blot may contain a series of normal individuals and a series ofcancer cases, tumors, or both. Southern blots displaying hybridizingfragments (differing in length from control DNA when probed withsequences near or including the EPHB2 locus) indicate a possiblemutation. If restriction enzymes that produce very large restrictionfragments are used, then pulsed field gel electrophoresis (PFGE) can beemployed.

A more definitive test for mutations in a candidate locus is to directlycompare genomic EPHB2 sequences from a person of interest with thosefrom a control population. In many instances, the control populationconsists of disease-free humans. The control population can also becomposed of patients who have the cancer being investigated. The wildtype genomic EPHB2 sequence can be obtained from the Entrez human genomesequence database (National Center for Biotechnology Information,Bethesda, Md.) under accession number NT_(—)004610. Alternatively, onecould sequence messenger RNA after amplification, thereby eliminatingthe necessity of determining the exon structure of the candidate gene.Suitable amplification methods include, but are not limited to, PCR,ligase chain reaction, Qbeta replicase, and isothermal amplification(e.g., strand displacement amplification).

Amplification Refractory Mutation System (ARMS) can also be used, asdisclosed in European Patent Application Publication No. 0332435 and inNewton et al, NUCL. ACIDS RES., 17:2503-2516 (1989). In addition,insertions and deletions of genes can be detected by cloning, sequencingand amplification. Furthermore, restriction fragment length polymorphism(RFLP) probes for the gene or surrounding marker genes can be used toscore alteration of an allele or an insertion in a polymorphic fragment.Such a method is useful for screening relatives of an affectedindividual for the presence of the EPHB2 mutation found in thatindividual.

Moreover, DNA microchip technology can be used. In a typical DNAmicrochip, a number of distinct oligonucleotide probes are built up inan array on a silicon chip. Nucleic acid to be analyzed is fluorescentlylabeled and hybridized to the probes on the chip. It is also possible tostudy nucleic acid-protein interactions using these nucleic acidmicrochips. Using this technique one can determine the presence ofmutations, or sequence the nucleic acid being analyzed. One can alsomeasure expression levels of a gene of interest. A microchip-basedmethod is one of parallel processing of many probes at once, and cantherefore significantly increase the rate of analysis.

Mutations from a person of interest falling outside the coding region ofEPHB2 can be detected by examining the non-coding regions, such asintrons and regulatory sequences. An early indication that mutations innoncoding regions are important may come from Northern blot experimentsthat reveal messenger RNA molecules of abnormal size or abundance in aperson of interest as compared to disease-free individuals.

Methods described in U.S. Patent Application Publication No. 20030022215can also be used for detecting the presence or absence of EPHB2 genealterations. The entire content of this publication is incorporatedherein by reference.

In order to detect EPHB2 gene alteration(s) in a tissue, it is helpfulto isolate the tissue free from surrounding tissues. Methods ofenriching a tissue preparation for specified types of cells are known inthe art. For example, the tissue may be isolated from paraffin orcryostat sections, and desired cells are separated from other cells byflow cytometry. These techniques, as well as other techniques forseparating one type of cells from other types, are well known in theart.

In another embodiment, alterations in the EPHB2 gene are detected byassessing the expression level or sequence of EPHB2 mRNA. Any techniqueknown in the art may be used for this purpose. These include, but arenot limited to, PCR amplification (including quantitative PCR orRT-PCR), Northern blot analysis, RNase protection, and DNA microarrays.Diminished mRNA expression may indicate an alteration in the EPHB2 gene.For example, a mutation in the promoter region may decrease the level oftranscription of the EPHB2 gene. For another example, a nonsensemutation can trigger rapid degradation of the RNA transcript via thenonsense mediated decay mechanism. A mutation in the 3′ untranslatedregion may also reduce the stability, and therefore the cellular level,of the affected mRNA transcript.

In one example, relative quantitative RT-PCR is used to amplify ordetect the level of the EPHB2 mRNA transcript(s). Reverse transcription(RT) of RNA to cDNA followed by relative quantitative PCR (RT-PCR) candetermine the relative concentrations of specific mRNA species isolatedfrom a person of interest. By determining that the concentration of aspecific mRNA species varies, it is shown that the gene encoding thespecific mRNA species is differentially expressed. In PCR, the number ofmolecules of the amplified target DNA increase by a factor approachingtwo with every cycle of the reaction until some reagent becomeslimiting. Thereafter, the rate of amplification becomes increasinglydiminished until there is no increase in the amplified target betweencycles. If a graph is plotted in which the cycle number is on the X-axisand the log of the concentration of the amplified target DNA is on theY-axis, a curved line of characteristic shape is formed by connectingthe plotted points. Beginning with the first cycle, the slope of theline is positive and constant. This is said to be the linear portion ofthe curve. After a reagent becomes limiting, the slope of the linebegins to decrease and eventually becomes zero. At this point theconcentration of the amplified target DNA becomes asymptotic to somefixed value. This is said to be the plateau portion of the curve.

The concentration of the target DNA in the linear portion of the PCRamplification is directly proportional to the starting concentration ofthe target before the reaction began. By determining the concentrationof the amplified products of the target DNA in PCR reactions that havecompleted the same number of cycles and are in their linear ranges, itis possible to determine the relative concentrations of the specifictarget sequence in the original DNA mixture. If the DNA mixtures arecDNAs synthesized from RNAs isolated from different tissues or samples,the relative abundances of the specific mRNA from which the targetsequence was derived can be determined for the respective tissues orsamples. This direct proportionality between the concentration of thePCR products and the relative mRNA abundances is true in the linearrange of the PCR reaction.

The final concentration of the target DNA in the plateau portion of thecurve is determined by the availability of reagents in the reaction mixand is independent of the original concentration of target DNA.Therefore, the first condition that must be met before the relativeabundances of an mRNA species can be determined by RT-PCR for acollection of RNA populations is that the concentrations of theamplified PCR products must be sampled when the PCR reactions are in thelinear portion of their curves.

The second condition that must be met for an RT-PCR experiment tosuccessfully determine the relative abundances of a particular mRNAspecies is that relative concentrations of the amplifiable cDNAs must benormalized to some independent standard. The goal of an RTPCR experimentis to determine the abundance of a particular mRNA species relative tothe average abundance of all mRNA species in the sample. Thus, in manycases, external and internal standards are used, to which the relativeabundance of other mRNAs are compared.

Many protocols for RT-PCR utilize internal PCR standards that areapproximately as abundant as the target. These strategies are effectiveif the products of the PCR amplifications are sampled during theirlinear phases. If the products are sampled when the reactions areapproaching the plateau phase, then the less abundant product becomesrelatively overrepresented. Comparisons of relative abundances made formany different RNA samples, such as is the case when examining RNAsamples for differential expression, become distorted in such a way asto make differences in relative abundances of RNAs appear less than theyactually are. This is not a significant problem if the internal standardis much more abundant than the target. If the internal standard is moreabundant than the target, then direct linear comparisons can be madebetween RNA samples.

The above discussion describes theoretical considerations for an RT-PCRassay for clinically derived materials. The problems inherent inclinical samples are that they are of variable quantity (makingnormalization problematic), and that they are of variable quality(necessitating the co-amplification of a reliable internal control,preferably of larger size than the target). Both of these problems areovercome if the RT-PCR is performed as a relative quantitative RT-PCRwith an internal standard in which the internal standard is anamplifiable cDNA fragment that is larger than the target cDNA fragmentand in which the abundance of the mRNA encoding the internal standard isroughly 5-100 fold higher than the mRNA encoding the target. This assaymeasures relative abundance, not absolute abundance of the respectivemRNA species.

Alterations in EPHB2 can also be detected by screening for alterationsin the structure or function of EphB2 proteins. For example, monoclonalantibodies immunoreactive with EphB2 can be used to screen a tissue.Lack of cognate antigen would indicate an EphB2 mutation. Antibodiesspecific for products of mutant alleles can also be used to detectmutant EPHB2 gene products. Such immunological assays can be done in anyconvenient formats known in the art. They can be competitive ornon-competitive, direct or indirect, and in the forward, reverse, orsimultaneous modes. Examples of suitable immunoassays include, but arenot limited to, latex or other particle agglutination,electrochemiluminescence, ELISAs, RIAs, sandwich or immunometric assays,time-resolved fluorescence, lateral flow assays, fluorescencepolarization, flow cytometry, immunohistochemical assays, Western blots,and proteomic chips. Those of skill in the art will know, or can readilydiscern, other suitable immunoassay formats without undueexperimentation.

The antibodies employed in the present invention can be used in liquidphase or bound to a solid phase carrier. Many solid carriers are suitedfor this purpose. Examples of these carriers include, but are notlimited to, glass, polystyrene, polypropylene, polyethylene, dextran,nylon, amylases, natural and modified celluloses, polyacrylamides,agaroses, or magnetite. The nature of the carrier can be either solubleor insoluble. In one example, antibodies are bound to solid phasecarriers by adsorption from an aqueous medium, although other modes ofaffixation, such as covalent coupling or other well-known means ofaffixation to the solid matrix can be used. Antibody molecules can bebound to a support before forming an immunocomplex with antigen. Theimmunocomplex can also be formed prior to binding the complex to thesolid support. Non-specific protein binding sites on the surface of thesolid phase support can be blocked. In one example, after adsorption ofsolid phase-bound antibodies, an aqueous solution of a protein free frominterference with the assay such as bovine, horse, or other serumalbumin can be admixed with the solid phase to adsorb the admixedprotein onto the surface of the antibody-containing solid support atprotein binding sites oil the surface that are not occupied by antibodymolecules.

Detection can be facilitated by coupling the antibody to a detectablesubstance. Examples of detectable substances include, withoutlimitation, enzymes, prosthetic groups, fluorescent materials,luminescent materials, bioluminescent materials, radioactive materials,and colloidal metals. Examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, galactosidase, oracetylcholinesterase. Examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin. Examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin. Examples of a luminescent material includeluminal. Examples of bioluminescent materials include luciferase,luciferin, and aequorin. Examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S, and ³H.

Another labeling technique that may result in greater sensitivityincludes coupling the antibodies to low molecular weight haptens. Thesehaptens can then be specifically detected by means of a second reaction.For example, it is common to use haptens such as biotin, which reactswith avidin, or dinitrophenol, pyridoxal, or fluorescein, which canreact with specific anti-hapten antibodies.

Antibodies employed in the present invention can be prepared by anyconventional method. These antibodies can be polyclonal, monoclonal,mono-specific, polyspecific, non-specific, humanized, human,single-chain, chimeric, synthetic, recombinant, hybrid, mutated,grafted, or in vitro generated antibodies. They can also be Fab,F(ab′₂), Fv, scFv, Fd, dAb, or other antibody fragments that retain theantigen-binding function. Preferably, an antibody of the presentinvention has an antigen-binding affinity of at least 10⁻⁵ M⁻¹, 10⁻⁶M⁻¹, 10⁻⁷ M⁻¹, 10⁻⁸ M⁻¹, 10⁻⁹ M⁻¹, or stronger. In one embodiment, anantibody of the present invention specifically recognizes a mutatedEphB2 protein sequence (such as an EphB2 mutant selected from Table 1 orTable 4) but not the wild type EphB2 sequence. The epitope on themutated EphB2 protein that is recognizable by the antibody may includeone or more point mutations (e.g., amino acid substitution, deletion, orinsertion). The epitope may also include altered post-translationalmodifications, or substitution, deletion or insertion of an amino acidsequence fragment. In addition, the epitope may include a new terminusgenerated by a nonsense mutation or other types of truncations. In manycases, an antibody specific for a mutated EphB2 protein has aninsignificant binding affinity to the wild type EphB2 protein, such asless than 10⁻⁴ M⁻¹, 10⁻³ M⁻¹, 10⁻² M⁻¹, 10⁻¹ M⁻¹ or weaker. In manyother cases, the binding affinity of the antibody to the mutated EphB2protein is at least 10, 10², 10³, 10⁴, 10⁵, or more times higher thanthat to the wild type EphB2 protein. In another embodiment, an antibodyof the present invention specifically recognizes the wild type EphB2protein sequence but not the mutated EphB2 sequences.

To prepare antibodies specific for mutated EphB2 proteins, EphB2peptides including the mutations can be prepared by using eitherrecombinant expression or chemical synthesis. Theses peptides mayinclude, without limitation, about 6-20 amino acid residues. Thepeptides can be conjugated with .ah immunogenic carrier or mixed with anadjuvant, such as Freund's complete or incomplete adjuvant, and theninjected into an animal to induce an antiEphB2 antibody response.Polyclonal antibodies specific for mutated EphB2 protein can be preparedusing affinity chromatography, in which antibody fractions that do notbind to the wild type sequences are poured into another affinity columncoupled with the mutated EphB2 peptide. Antibodies retained by thecolumn are eluted and tested for their specificity for mutated EphB2versus the wild type sequence.

Monoclonal antibodies specific for the mutated EphB2 sequence can alsobe prepared by using standard techniques. Briefly, an immortal cell line(typically a myeloma) is fused to lymphocytes (typically splenocytes)from a mammal immunized with an EphB2 immunogen carrying the mutation,and the culture supernatants of the resulting hybridoma cells arescreened to identify a hybridoma producing a monoclonal antibody thatspecifically binds to the mutated EphB2 sequence, but not the wild typesequence.

Other methods suitable for detecting alterations in EphB2 proteinsinclude, but are not limited to, two-dimensional gel electrophoresis,mass spectrometry, or other high-throughput polypeptide sequencing oridentification methods. In addition, functional assays can be used. Forexample, it is known that EphB2 proteins bind specifically to EFNB.Thus, an assay for this binding activity can be employed to detectmutations in EphB2 proteins. For another example, EphB2 proteins candown-regulate the RAS-MAPK pathway. Consequently, an assay for theRAS-MAPK pathway can be used to evaluate the biological function ofEphB2 proteins. Abnormalities in EphB2 proteins indicate alterations inthe EPHB2 gene.

The present invention also contemplates antibodies specificallyrecognizing EphB2 proteins. In one embodiment, the present inventionprovides a monoclonal antibody specifically recognizing amino acids118-137 of SEQ ID NOs: 1 or 3. Such an antibody can be prepared bycoupling a peptide consisting of amino acid 118-137 of SEQ ID NOs: 1 or3 to a carrier for immunizations. A cysteine residue can be added to theC-terminus of the peptide to facilitate its conjugation with thecarrier.

Moreover, the present Invention features methods for detectingalterations or abnormalities in EphB2-mediated or -associated signaltransduction pathways. Abnormalities in the EphB2-mediated signaltransduction pathways can lead to reduced or abolished tumor suppressionactivity, thereby leading to the development of cancer. Theseabnormalities are therefore indicative of the risk, predisposition,disease status, or staging of cancer. Assays suitable for evaluatingEphB2-mediated or -associated signal transduction pathways include, butare not limited to, those described in Paraskevas et al, FEBS LETT,455:203-208 (1999); Boucher et al, J CELL B IOCHEM, 79:355-369 (2000);Giehl et al, ONCOGENE, 19:4531-41 (2000); Ellenrieder et al, CANCER RES,61:4222-4228 (2001); Woods et al, MOL CELL BIOL, 21:3192-3205 (2001);Murphy et al, BR J CANCER, 84:926-35 (2001); Ryder et al, J CELLPHYSIOL, 186:53-64 (2001); Yip-Schneider et al, BIOCHEM BIOPHYS RESCOMMUN, 280:992-997 (2001); Ding and Adrian, BIOCHEM BIOPHYS RES.COMMUN, 282:447-453 (2001); Takasu et al, SCIENCE, 295:491-495 (2002);Murai and Pasquale, NEURON, 33:159-162 (2002); and Irie and Yamaguchi,NATURE NEUROSCI, 5:1117-1118 (2002), all of which are incorporatedherein by reference in their entireties.

In addition, the present invention features methods for detectingabnormalities in the expression or function of the EPHB2 gene that arecaused not by alterations in the EPHB2 gene but by changes in thestructures or functions of other genes. Abnormalities in the upstreamregulators of EPHB2 can lead to the dysfunction of the EPHB2 gene and,therefore, increase the risk of cancer. Examples of these upstreamregulators include, but are not limited to, Beta-catenin and TCF. See,e.g., Batlle, et al, CELL, 111:251-263 (2002). The function of EphB2protein can also be impaired by inappropriate protein folding orpost-translational modification due to defects in a protein processingenzyme. Moreover, the dysfunction of an EphB2 effector may lead tocancer. Accordingly, like mutations in the EPHB2 gene, abnormalities inEPHB2 regulators/effectors can also be suggestive of the presence,staging, or risk of cancer.

Furthermore, the present invention provides methods for selectingpersonalized therapies based on the presence or absence of alterationsin the EPHB2 gene or its associated pathways. For example, a subjecthaving a mutation or abnormality in the EPHB2 gene or its associatedpathways is more likely to be responsive to an EPHB2 pathway-directedtreatment than a subject who does not have any EPHB2 mutation. Thus, bydetecting alterations in the EPHB2 gene or its related pathways, one canidentify patients who are likely to benefit from EPHB2-specifictherapies.

The EPHB2 gene or its associated pathways can also be used to monitorthe effect or efficacy of an anti-cancer treatment. For instance, theefficacy of an anti-cancer treatment can be evaluated based on whetherthe treatment restores or decreases an abnormality in the EPHB2 gene oran EPHB2-related pathway.

Cancers that are amenable to the present invention include, but are notlimited to, prostate cancer, breast cancer, brain cancer (e.g.,neuroblastoma, glioblastomas, medulloblastoma, astrocytoma,oligodendroglioma, ependymomas), lung cancer, liver cancer, spleencancer, kidney cancer, pancreas cancer, intestine cancer, leukemia,lymphoma, colon cancer, uterus and endometrium cancer, cervix cancer,stomach cancer, testicle cancer, ovary cancer, skin cancer, head andneck cancer, esophagus cancer, or other cancers in which EPHB2 or itsassociated pathways play a role in tumorigenesis. The methods of thepresent invention can be used to assess the presence, predisposition, orrisk of cancer in humans as well as animals (e.g., dogs, cats, or otherdomesticated mammals).

III. THERAPEUTIC AND PROPHYLACTIC TREATMENTS

The present invention also features compositions and methods for thetreatment or prevention of cancers. In many embodiments, thecompositions of the present invention are capable of correcting oralleviating an abnormality in the EPHB2 gene or an EPHB2-relatedsignaling pathway. In one example, a composition of the presentinvention includes a wild type EphB2 protein, or a biologically activefragment, variant or mimic thereof. As used herein, “biologicallyactive” refers to the ability to correct or reduce an abnormalityassociated with an impaired EPHB2 gene or its expression product(s). Inanother example, a composition of the present invention includes anexpression vector or a gene delivery vector that encodes a wild typeEphB2 protein or a biologically active fragment, variant or mimicthereof.

In many other embodiments, the compositions of the present invention cankill or induce apoptosis in cancer cells. However, for treating a tumor,it is not necessary that the tumor cell be killed or induced to undergoapoptosis. Rather, to accomplish a meaningful treatment or prevention,all that is required for a composition of the present invention is thatthe tumor growth be slowed to some degree or the development of cancerbe delayed. It may be that the tumor growth/development is completelyblocked or averted, however, or that some tumor regression is achieved.

Protein Therapy

In one aspect, the present invention features administration of atherapeutically or prophylactically effective amount of an EphB2polypeptide, or a biologically active fragment, variant or mimicthereof, to a subject in need thereof. Formulations including an EphB2polypeptide (or a biologically active derivative thereof) can beprepared based on the route of administration and purpose. Suitableformulations include, but are not limited to, liposomal formulations andother classic pharmaceutical preparations.

In many cases, an EphB2 polypeptide, or a biologically active derivativethereof, is prepared in an isolated or purified form. Such a preparationis substantially free from other proteins, or contains only aninsignificant amount of contaminants that would not interfere with=theintended use of the preparation.

An EphB2 protein (e.g., variants 1 or 2 depicted in SEQ ID NOs: 1 or 3,respectively) can be prepared by any method known in the art, such asrecombinant expression technology or chemical synthesis. In oneembodiment, an EphB2 protein is produced by expressing an expressionvector in host cells. The expression vector includes an expressioncontrol sequence operably linked to a codon sequence that encodes theEphB2 protein. The expression vector can be introduced into the hostcells by transfection, transformation, or transduction.

Suitable host cells include mammalian cells, insect cells, yeast, orbacteria. Other eukaryotic or prokaryotic cells can also be used.Specific examples of suitable host cells include, but are not limitedto, Chinese hamster ovary cells (CHO), HeLa cells, COS cells, 293 cells,CV-1 cells, E. coli (e.g., HB101, MC1061), B. subtilis, and Pseudomonas.In addition to cell lines, the host cells can also be primary cellcultures. Cells in transgenic animals or plants can also be used toproduce EphB2 proteins. The selection of suitable host cells and methodsfor culture, transfection/transformation, amplification, screening,product production, and purification are well known in the art.

The present invention also features biologically active variants of anEphB2 protein. These variants retain at least a substantial portion ofthe biological activity of the original EphB2 protein, and are capableof reversing or alleviating the cellular abnormalities caused byinactivation of the EPHB2 gene. In one example, a variant of an EphB2protein retains at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% ofthe biological activity of the original protein. In another example, avariant of an EphB2 protein shows an increased biological activitycompared to the original protein. The biological activity of an EphB2protein or its variant can be determined by any method known in the art.Exemplary methods include, but are not limited to, the cell growth assayillustrated in FIG. 4A and the colony formation assay depicted in FIG.4B.

In many embodiments, the amino acid sequence of a variant issubstantially identical to that of the original protein. For instance, avariant can share at least 80%, 85%, 90%, 95%, or 99% global sequenceidentity with the original protein. Sequence identity or similarity canbe determined by a variety of methods including, but not limited to,those described in Latched et al, J MOL BIOL, 215:403-410 (1990),Needleman et al, J MOL BIOL, 48:444-453 (1970), and Meyers et al,CABIOS, 4:11-17 (1988). Computer programs suitable for this purposeinclude the BLAST programs provided by NCBI, MegAlign provided byDNASTAR (Madison, Wis.), and the GAP program provided by the GeneticsComputer Group (GCG). For the GAP program, default values may be used(e.g., the penalty for opening a gap in one of the sequences is 11 andfor extending the gap is 8).

A variant of an EphB2 protein can be naturally occurring, such as byallelic variation or polymorphism. It can also be deliberatelyengineered. In addition, a variant of a human EphB2 protein can be aspecies homologue of the human protein, such as a murine or rat EphB2.An EphB2 variant can be prepared from the original protein through aminoacid additions, deletions, substitutions, or other modifications.Methods suitable for this purpose include, but are not limited to,recombinant DNA technology or chemical synthesis (including solid phasesynthesis). The amino acid residue(s) in the original peptide can alsobe modified by adding a polysaccharide, lipid, or other moiety toenhance the binding specificity or affinity, or the stability of theprotein.

Substitutional variants typically contain the exchange of one amino acidfor another at one or more sites within the protein, and may be designedto modulate one or more properties of the polypeptide, such as stabilityagainst proteolytic cleavage, without the loss of other functions orproperties. Amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involved.Preferred substitutions are conservative, that is, one amino acid isreplaced with one of similar shape and charge. Conservativesubstitutions are well known in the art. Exemplary conservativesubstitutions include substitutions within the following groups: (1)glycine and alanine; (2) valine, isoleucine, and leucine; (3) asparticacid and glutamic acid; (4) asparagine and glutamine; (5) serine andthreonine; (6) lysine and arginine; and (7) tyrosine and phenylalanine.The use of the hydrophobic index or hydrophilicity in designingpolypeptides is discussed in U.S. Pat. No. 5,691,198. In one example, avariant is derived from the original protein by at least 1, 2, 3, 4, 5,10, 20, or more amino acid substitutions.

In addition, the present invention features mimics of an EphB2 proteinor a biologically active fragment or variant thereof. A protein mimicretains many of the biologically important structural features of theparent protein while differing from the parent protein in many othersignificant ways. The underlying rationale behind the use of proteinmimics is that the peptide backbone of proteins exists chiefly to orientamino acid side chains in such a way as to facilitate molecularinteractions, such as those of antibody and antigen, enzyme andsubstrate or scaffolding proteins. A protein mimetic is designed topermit molecular interactions similar to the natural molecule.

In one example, the amide bonds in an EphB2 protein (or a biologicallyactive fragment thereof) is replaced with non-peptidic constraints thatbring drug-like properties like stability and bioavailability to themolecule. In another example, a chemical compound that mimics thestructure of an EphB2 fragment is identified or rationally designed. Avariety of methods are available for determining the structure of apeptide fragment. These methods include, but are not limited to, X-raycrystallography and NMR. Similar techniques can be used to analyze theinteraction interface between an EphB2 protein and its binding orinteraction partner. Once a three-dimensional structure of the EphB2protein is obtained, a large number of compounds can be analyzed bycomputer programs to identify those that mimic the structure or actionof the EphB2 protein at the interaction interface. A compound soselected can be further fine tuned or optimized to generate a drug ordrug candidate.

An EphB2 protein or a biologically active variant thereof can be furtherconjugated or fused to another polypeptide or carrier by usingconventional techniques. Many of these carriers afford the conjugatedmolecule with improved stability, increased bioavailability, or reducedimmunogenicity. The conjugation may be covalent or non-covalent.

In one example, an EphB2 protein (or a biologically active derivativetherefore) is fused with an Fc fragment. Preferably, the Fc fragment isnot immunogenic to the subject being treated. In another example, anEphB2 protein (or a biologically active derivative thereof) isconjugated to a non-protein macromolecular carrier. Such amacromolecular carrier can include, for example, lipid-fatty acidconjugates, polyethylene glycol, or carbohydrates.

Nucleic Acid Based Therapies

Another therapy approach employed by the present invention is toprovide, to a cancer cell, an expression vector that encodes a wild typeEphB2 or a biologically active variant thereof. Preferred vectors forthis purpose include, but are not limited to, viral vectors such asretroviral, lentiviral, adenoviral, adeno-associated viral (AAV), herpesviral, alphavirus, astrovirus, coronavirus, orthomyxovirus, papovavirus,paramyxovirus, parvovirus, picornavirus, poxvirus, or togavirus vectors.Also preferred are liposomally encapsulated expression vectors.

Those of skill in the art are well aware of how to apply gene deliveryto in vivo and ex vivo situations. For viral vectors, one generally willprepare a viral vector stock. Depending on the kind of virus and thetiter attainable, one will deliver 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸,1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹² or more infectious particles to a subjectof interest. Similar figures may be extrapolated for liposomal or othernon-viral formulations by comparing relative uptake efficiencies.Formulation as a pharmaceutically acceptable composition is discussedbelow.

Various routes are contemplated for various tumor types. For practicallyany tumor, systemic delivery is contemplated. This will prove especiallyimportant in attacking microscopic or metastatic cancer. Where discretetumor mass may be identified, a variety of direct, local and regionalapproaches may be taken. For example, the tumor may be directly injectedwith the expression vector. A tumor bed may be treated prior to, duringor after resection. Following resection, one may deliver the vector by acatheter left in place following surgery. One may also utilize the tumorvasculature to introduce the vector into the tumor by injecting asupporting vein or artery. A more distal blood supply route also may beutilized.

In a different embodiment, ex vivo gene therapy is contemplated. Thisapproach is particularly suited, although not limited, to treatment ofbone marrow associated cancers. In an ex vivo embodiment, cells from thepatient are removed and maintained outside the body for at least someperiod of time. During this period, a therapy is delivered, after whichthe cells are reintroduced into the patient; hopefully, any tumor cellsin the sample have been killed.

Autologous bone marrow transplant (ABMT) is an example of ex vivo genetherapy. Basically, the notion behind ABMT is that the patient willserve as his or her own bone marrow donor. Thus, a normally lethal doseof irradiation or chemotherapeutic may be delivered to the patient tokill tumor cells, and the bone marrow repopulated with the patients owncells that have been maintained (and perhaps expanded) ex vivo. Because,bone marrow often is contaminated with tumor cells, it is desirable topurge the bone marrow of these cells.

The present invention also features antisense or RNA interference (RNAi)sequences that can specifically inhibit the transcription or translationof mutated EPHB2 genes (e.g., those depicted in Table 1) but not thewild type EPHB2 gene. In addition, the present invention contemplatesvectors comprising or encoding an antisense or RNAi sequence of thepresent invention. Antisense or RNAi sequences are useful for inhibitingor alleviating abnormally high expression or activity of the EPHB2 genecaused by mutations or alterations in the gene or its associatedsignaling pathways.

An antisense polynucleotide comprises a nucleotide sequencecomplementary to a sense polynucleotide, e.g. complementary to thecoding strand of a double-stranded cDNA molecule or to an mRNA sequence.An antisense polynucleotide can be complementary to the entire codingstrand of EPHB2, or a portion thereof. An antisense polynucleotidemolecule can also be complementary to a noncoding region of the codingstrand of EPHB2.

In many embodiments, an antisense polynucleotide of the presentinvention includes at least about 10, 15, 20, 25, 30, 35, 40, 45, 50 ormore nucleotides. An antisense polynucleotide can be designed accordingto the rules of Watson and Crick base pairing. In one embodiment, anantisense polynucleotide is chemically synthesized using naturallyoccurring nucleotides or variously modified nucleotides designed toincrease the biological stability of the molecules or to increase thephysical stability of the duplex formed between the antisense and sensepolynucleotides. Examples of modified nucleotides which can be used togenerate an antisense polynucleotide include 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxymethyl) uracil,5carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladen4exine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl,2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, and 2,6-diaminopurine. In another embodiment, anantisense polynucleotide of the present invention is producedbiologically by using an expression vector into which the targetsequence is subcloned in an antisense orientation.

RNAi is a technique for post-transcriptional gene silencing (“PTGS”), inwhich target gene activity is specifically abolished with cognatedouble-stranded RNA (“dsRNA”). RNAi resembles in many aspects PTGS inplants and has been detected in many invertebrates includingtrypanosome, hydra, planaria, nematode and fruit fly (Drosophilamelanogaster). It may be involved in the modulation of transposableelement mobilization and antiviral state formation. In mammalian cells,introduction of long dsRNA can initiate a potent antiviral response,exemplified by nonspecific inhibition of protein synthesis and RNAdegradation. However, when short dsRNA, homologous to the target gene,is introduced into the cell, a sequence specific reduction in the targetgene activity is observed. RNA interference provides a mechanism of genesilencing at the mRNA level.

Sequences capable of inhibiting gene expression by RNA interference canhave any desired length. For instance, the sequence can have at least15, 20, 25, or more consecutive nucleotides. The sequence can be dsRNAor any other type of polynucleotide, provided that the sequence can forma functional silencing complex to degrade the target mRNA transcript.Examples of suitable RNAi sequences include, but are not limited to,short interfering RNAs, short hairpin RNAs, microRNAs, or smallmodulatory RNAs. See, for example, Novina and Sharp, NATURE, 430:161-164(2004).

Immunotherapies

Immunotherapeutics, generally, rely on the use of immune effector cellsor molecules to target and destroy cancer cells. The immune effector maybe, for example, an antibody specific for the mutated EphB2 protein orother markers on the surface of a tumor cell. The antibody alone mayserve as an effector of therapy or it may recruit other cells toactually effect cell killing. The antibody also may be conjugated to adrug or toxin (chemotherapeutic, radionuclide, ricin A chain, choleratoxin, pertussis toxin, etc.) and serve' as a targeting agent.Alternatively, the effector may be a lymphocyte carrying a surfacemolecule that interacts, either directly or indirectly, with a tumorcell target, such as a mutated EphB2 protein. Various effector cellsinclude cytotoxic T cells and NK cells.

An immunotherapy can also be used as part of a combined therapy, forinstance, in conjunction with EPHB2-targeted gene therapy. The generalapproach for combined therapy is discussed below.

In one embodiment, the present invention provides immunoconjugates orimmunotoxins for the prevention or treatment of cancers. Animmunoconjugate or immunotoxin of the present invention comprises anantibody capable of binding to a cancer marker (such as a mutant EphB2)and linked to a cytotoxic or anticellular agent. The cytotoxic oranticellular agent has the ability to kill or suppress the growth ordivision of cells.

Exemplary anticellular agents include, but are not limited to,chemotherapeutic agents, radioisotopes, and cytotoxins. Example ofchemotherapeutic agents include hormones such as steroids;antimetabolites such as cytosine arabinoside, fluorouracil, methotrexateor aminopterin; anthracycline; mitomycin C; vinca alkaloids;demecolcine; etoposide; mithramycin; or alkylating agents such aschlorambucil or melphalan.

Preferred immunotoxins often include, to mention just a few examples, aplant-, fungal- or bacterial-derived toxin, such as an A chain toxin, aribosome inactivating protein, asarcin, aspergillin, restirictocin, aribonuclease, diphtheria toxin, or pseudomonas exotoxin. Combinations ofvarious toxins can be coupled to one antibody molecule, therebyaccommodating variable or even enhanced cytotoxicity.

The preparation of immunotoxins is, in general, well known .in the art(see, e.g., U.S. Pat. No. 4,340,535, which is incorporated herein byreference). It also is known that while IgG based immunotoxins mayexhibit better binding capability and slower blood clearance than theirFab′ counterparts, Fab′ fragment-based immunotoxins can exhibit bettertissue penetrating capability as compared to IgG based immunotoxins.

One type of toxin for attachment to antibodies is ricin, withdeglycosylated ricin A chain being particularly preferred. As usedherein, the term “ricin” is intended to refer to ricin prepared fromboth natural sources and by recombinant means. Various recombinant orgenetically engineered forms of the ricin molecule are known to those ofskill in the art, all of which may be employed in accordance with thepresent invention:

Deglycosylated ricin A chain (dgA) is preferred because of its extremepotency, longer half-life, and because it is economically feasible tomanufacture it a clinical grade and scale (available commercially fromInland Laboratories, Austin, Tex.). Truncated ricin A chain, from whichthe 30 N-terminal amino acids have been removed by Nagarase (Sigma),also may be employed.

Linking or coupling one or more toxin moieties to an antibody may beachieved by a variety of mechanisms, for example, covalent binding,affinity binding, intercalation, coordinate binding and complexation.Preferred binding methods are those involving covalent binding, such asusing chemical cross-linkers, natural peptides or disulfide bonds.

The covalent binding can be achieved either by direct condensation ofexisting side chains or by the incorporation of external bridgingmolecules. Many bivalent or polyvalent agents are useful in couplingprotein molecules to other proteins, peptides or amine functions.Examples of coupling agents are carbodiimides, diisocyanates,glutaraldehyde, diazobenzenes, and hexamethylene diamines. This list isnot intended to be exhaustive of the various coupling agents known inthe art but, rather, is exemplary of the more common coupling agentsthat may be used.

In many embodiments, it is contemplated that one may wish to firstderivatize the antibody, and then attach the toxin component to thederivatized product. As used herein, the term “derivatize” is used todescribe the chemical modification of the antibody substrate with asuitable cross-linking agent. Examples of cross-linking agents for usein this manner include the disulfide-bond containing linkers SPDP(N-succinimidyl-3-(2-pyridyldithio)propionate) and SMPT(4-succinimidyl-oxycarbonyl-a-methyl-a(2-pyridyldithio)toluene).

Biologically releasable bonds can also be employed in constructing aclinically active immunotoxin, such that the toxin moiety is capable ofbeing released from the antibody once it has entered the target cell.Numerous types of linking constructs are known, including simply directdisulfide bond formation between sulfhydryl groups contained on aminoacids such as cysteine, or otherwise introduced into respective proteinstructures, and disulfide linkages using available or designed linkermoieties.

Numerous types of disulfide-bond containing linkers are known which cansuccessfully be employed to conjugate toxin moieties to antibodies.Certain linkers are preferred, such as, for example, sterically hindereddisulfide bond linkers are preferred due to their greater stability invivo, thus preventing release of the toxin moiety prior to binding atthe site of action. Another preferred cross-linking reagent is SMPT,although other linkers such as SATA, SPDP and 2-iminothiolane also maybe employed.

Once conjugated, the conjugate can be purified to remove contaminantssuch as unconjugated A chain or antibody. In many cases, it is importantto remove unconjugated A chain because of the possibility of increasedtoxicity. Moreover, unconjugated antibody may be removed to avoid thepossibility of competition for the antigen between conjugated andunconjugated species. Numerous purification techniques can be used toprovide conjugates to a sufficient degree of purity to render themclinically useful.

After a sufficiently purified conjugate has been prepared, one maydesire to prepare it into a pharmaceutical composition that may beadministered parenterally. This can be done, for example, by using forthe last purification step a medium with a suitable pharmaceuticalcomposition. Such formulations will typically include pharmaceuticalbuffers, along with excipients, stabilizing agents and such like. Thepharmaceutically acceptable compositions are often sterile,non-immunogenic and non-pyrogenic. Details of their preparation are wellknown in the art.

Suitable pharmaceutical compositions in accordance with the inventioncan comprise, without limitation, from about 10 to about 100 mg of thedesired conjugate admixed with an acceptable pharmaceutical diluent orexcipient, such as a sterile aqueous solution, to give a finalconcentration of about 0.25 to about 2.5 mg/ml with respect to theconjugate.

As mentioned above, the antibodies of the invention may be linked to oneor more chemotherapeutic agents, such as anti-tumor drugs, cytokines,antimetabolites, alkylating agents, hormones, nucleic acids and thelike, which may be targeted to cancer cell or cancer-prone cell thatexpresses mutated EphB2 proteins. The advantage of antibody-conjugatedagents over their non-antibody conjugated counterparts is the addedselectivity afforded by the antibody.

In analyzing the variety of chemotherapeutic and pharmacologic agentsavailable to conjugate to an antibody, one may wish to particularlyconsider those that have been previously shown to be successfullyconjugated to antibodies and to function pharmacologically. Exemplaryantineoplastic agents that have been used include doxorubicin,daunomycin, methotrexate, and vinblastine. Moreover, the attachment ofother agents such as neocarzinostatin, macromycin, trenimon anda-amanitin has also been described. The lists of suitable agentspresented herein are, of course, merely exemplary in that the technologyfor attaching pharmaceutical agents to antibodies for specific deliveryto tissues is well established.

Combined Therapy with Immunotherapy, Traditional Chemo- or Radiotherapy

Tumor cell resistance to DNA damaging agents represents a major problemin clinical oncology. One goal of current cancer research is to findways to improve the efficacy of chemo- and radiotherapy. One way is bycombining such traditional therapies with gene therapy, such as an EPHB2replacement therapy.

To kill cells, inhibit cell growth, inhibit metastasis, inhibitangiogenesis or otherwise reverse or reduce the malignant phenotype oftumor cells, one may contact a target cell with an EphB2 protein or anEPHB2 expression construct and at least one other agent. Thesecompositions would be provided in a combined amount effective to kill orinhibit proliferation of the cell. This process may involve contactingthe cells with the protein/expression construct and the agent(s) orfactor(s) at the same time. This may be achieved by contacting the cellwith a single composition or pharmacological formulation that includesboth agents, or by contacting\ the cell with two distinct compositionsor formulations at the same time, wherein one composition includes theexpression construct and the other includes the agent.

Alternatively, the gene/protein therapy treatment may precede or followthe other agent treatment by intervals ranging from minutes to weeks. Inembodiments where the other agent and the EphB2 protein/expressionconstruct are applied separately to the cell, one may ensure that asignificant period of time did not expire between the time of eachdelivery, such that the agent and expression construct would still beable to exert an advantageously combined effect on the cell. In suchinstances, it is contemplated that one would contact the cell with bothmodalities within about 12-24 hours of each other and, more preferably,within about 6-12 hours of each other, with a delay time of only about12 hours being most preferred. In some situations, it may be desirableto extend the time period for treatment significantly, however, whereseveral days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7or 8) lapse between the respective administrations.

It also is conceivable that more than one administration of either EphB2or the other agent will be desired. Various combinations may beemployed, where EphB2 is “A” and the other agent is “B,” as exemplifiedbelow:

A/BA, B/A/B, B/B/A, A/A/B, B/A/A, A/B/B, B/B/B/A, B/B/A/B, A/A/B/B,A/B/A/B, A/B/B/A, B/B/A/A, B/A/B/A, B/A/A/B, B/B/B/A, A/A/A/B, B/A/A/A,A/B/A/A, A/A/B/A, A/B/B/B, B/A/B/B, or B/B/A/B.

Other combinations are contemplated. Again, to achieve cell killing,both agents are delivered to a cell in a combined amount effective tokill the cell.

Agents or factors suitable for use in a combined therapy are anychemical compound or treatment method that induces DNA damage whenapplied to a cell. Such agents and factors include radiation and wavesthat induce DNA damage such as γ-irradiation, X-rays, UV-irradiation,microwaves, electronic emissions, and the like. A variety of chemicalcompounds, also described as chemotherapeutic agents, function to induceDNA damage, all of which are intended to be of use in the combinedtreatment methods disclosed herein. Chemotherapeutic agents contemplatedto be of use, include, e.g., adriamycin, 5-fluorouracil (5FU), etoposide(VP-16), camptothecin, actinomycin-D, mitomycin C, cisplatin (CDDP), andin some cases, hydrogen peroxide. The invention also encompasses the useof a combination of one or more DNA damaging agents, whetherradiation-based or actual compounds, such as the use of X-rays withcisplatin or the use of cisplatin with etoposide. In certainembodiments, the use of cisplatin in combination with an EphB2 proteinor an EphB2 expression construct is preferred.

The skilled artisan is directed to REMINGTON′S PHARMACEUTICAL SCIENCES(15^(th) Edition), chapter 33, in particular pages 624-652, the entirecontents of which are incorporated herein by reference. Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration willdetermine the appropriate dose for the individual subject.

The present invention contemplates the regional delivery of an EphB2protein or expression construct to patients with EPHB2-linked cancers.Similarly, the immunotherapy may be directed to a particular, affectedregion of the subject's body. Alternatively, systemic delivery of aprotein, expression construct or immunotoxin may be appropriate incertain circumstances, for example, where extensive metastasis hasoccurred.

Formulations and Routes for Administration to Patients

Where clinical applications are contemplated, it will be necessary toprepare pharmaceutical compositions which comprise expression vectors,virus stocks, proteins, antibodies or drugs in a form appropriate forthe intended application. In many instances, this will entail preparingcompositions that are essentially free of pyrogens, as well as otherimpurities that could be harmful to humans or animals.

A pharmaceutical composition of the present invention typically includesan active component (e.g., an EphB2 protein, an expression vectorencoding the same, or an antibody specific for a mutated EphB2 protein)and a pharmacologically acceptable carrier. As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents and the like. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the vectors or cells of the present invention, its usein therapeutic or prophylactic compositions is contemplated.Supplementary active ingredients also can be incorporated into thecompositions.

The active compositions of the present invention may include classicpharmaceutical preparations. Administration of these compositionsaccording to the present invention will be via any common route so longas the target tissue is available via that route. This includes oral,nasal, buccal, rectal, vaginal or topical. Alternatively, administrationmay be by orthotopic, intradermal, subcutaneous, intramuscular,intraperitoneal intratumoral, circumferentially, catheterization, orintravenous injection. Such compositions would normally be administeredas pharmaceutically acceptable compositions.

The active compounds may also be administered parenterally orintraperitoneally. Solutions of the active compounds as free base orpharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations may contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In most cases, the form is sterile and fluid to the extentthat easy syringability exists. It preferably is also stable under theconditions of manufacture and storage and preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof, orvegetable oils. The proper fluidity can be maintained, for example, bythe use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion, or by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial or antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, orthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousother ingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thevarious sterilized active ingredients into a sterile vehicle whichcontains the basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying or freeze-drying techniques that yield apowder of the active ingredient plus any additional desired ingredientfrom a previously sterile filtered solution thereof.

For oral administration, the polypeptides, nucleic acids or theiranalogs employed in the present invention can be incorporated withexcipients and used in the form of noningestible mouthwashes anddentifrices. A mouthwash may be prepared incorporating the activeingredient in the required amount in an appropriate solvent, such as asodium borate solution (Dobell's Solution). Alternatively, the activeingredient may be incorporated into an antiseptic wash containing sodiumborate, glycerin and potassium bicarbonate. The active ingredient mayalso be dispersed in dentifrices, including: gels, pastes, powders, orslurries. The active ingredient may be added in a therapeutically orprophylactically effective amount to a paste dentifrice that may includewater, binders, abrasives, flavoring agents, foaming agents, orhumectants.

The compositions of the present invention may be formulated in a neutralor salt form. Pharmaceutically acceptable salts include the acidaddition salts formed with the free amino groups of the protein) orformed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

Upon formulation, compositions or solutions will be administered in amanner compatible with the dosage formulation and in such amount as istherapeutically or prophylactically effective. The formulations areeasily administered in a variety of dosage forms such as injectablesolutions, drug release capsules and the like. For parenteraladministration in an aqueous solution, for example, the solution can besuitably buffered if necessary and the liquid diluent first renderedisotonic with sufficient saline or glucose. These particular aqueoussolutions are especially suitable for intravenous, intramuscular,subcutaneous, and intraperitoneal administration. In this connection,sterile aqueous media that can be employed will be known to those ofskill in the art in light of the present disclosure. For example, onedosage could be dissolved in 1 ml of isotonic NaCl solution and eitheradded to 1000 ml of hypodermoclysis fluid or injected at the proposedsite of infusion, (see, for example, REMINGTON′S PHARMACEUTICAL SCIENCES(15th Edition), pages 1035-1038 and 1570-1580). Some variation in dosagewill necessarily occur depending on the condition of the subject beingtreated.

IV. METHODS FOR SCREENING ACTIVE COMPOUNDS OR AGENTS FOR MUTATED EPHB2GENE

The present invention also contemplates the use of EphB2, fragmentsthereof, or nucleic acids coding the same in the screening of compoundsor agents for activities in either stimulating EphB2 activity,overcoming the lack of EphB2 activity, or blocking the effect of amutant EphB2 molecule. These assays may make use of a variety ofdifferent formats and may depend on the kind of activity for which thescreen is being conducted. Contemplated functional “read-outs” include,but are not limited to, binding to a compound/agent, inhibition ofbinding to a substrate, ligand, receptor or other binding partner by acompound/agent, inhibition or stimulation of an EphB2-mediated or-associated signaling pathway, growth, metastasis, cell division, cellmigration, soft agar colony formation, contact inhibition, invasiveness,angiogenesis, apoptosis, tumor progression, or other malignantphenotype.

In Vitro Assays

In one embodiment, the invention is to be applied for the screening ofcompounds/agents that bind to an EphB2 protein or a fragment thereof.The protein or fragment may be either free in solution, fixed to asupport, expressed in or on the surface of a cell. Either the protein orthe compound/agent may be labeled, thereby permitting the determinationof binding.

In another embodiment, the assay may measure the inhibition of bindingof EphB2 to a natural or artificial substrate or binding partner.Competitive binding assays can be performed in which one of the agents(EphB2, binding partner or compound) is labeled. Usually, the EphB2protein or its fragment will be the labeled species. One may measure theamount of free label versus bound label to determine binding orinhibition of binding.

Compound libraries and techniques for high throughput screening ofcompounds can be used. See, e.g., WO 84/03564. In one format, largenumbers of small peptide test compounds can be synthesized on a solidsubstrate, such as plastic pins or some other surface. The peptide testcompounds are reacted with EphB2 and washed. Bound polypeptide isdetected by various methods.

Purified EphB2 can be coated directly onto plates for use in theaforementioned drug screening techniques. However, non-neutralizingantibodies to the protein can be used to immobilize the EphB2 protein toa solid phase. Also, fusion proteins containing a reactive region(preferably a terminal region) may be used to link the EphB2 activeregion to a solid phase.

Various cell lines containing wild type or natural or engineeredmutations in EphB2 can be used to study various functional attributes ofEphB2 and how a candidate compound/agent affects these attributes. Manyof these naturally occurring mutations in EphB2 lead to, contribute to,or otherwise cause malignancy. Other mutations can be introduced intothe wild type EphB2 protein by standard genetic engineering techniques.In one assay format, a compound of interest is formulated appropriately,given its biochemical nature, and then contacted with a target cell.Depending on the assay, culture may be required. The cell may then beexamined by virtue of a number of different physiologic assays.Alternatively, molecular analysis may be performed in which the functionof EphB2; or related pathways, may be explored. This may involve assayssuch as those for protein expression, enzyme function, substrateutilization, phosphorylation states of various molecules includingEphB2, mRNA expression (including differential display of whole cell orpoly-A RNA), and others.

In one example, cells lacking functional EPHB2 gene or expressionproducts are prepared by using a variety of means, as appreciated bythose skilled in the art. The inactivation or deletion of the EPHB2 geneor its expression products can be permanent (e.g., via traditional geneknockout technology) or transient (e.g., via RNAi technology). Thesecells can be used to address the sensitivity of EPHB2−/− cells to drugtherapy. A typical method includes contacting the EPHB2−/− cells with anagent of interest, and comparing the phenotype or malignancy of thecells before and after said contact with the agent in order to determineif the agent is effective in overcoming cellular abnormalities caused bythe inactivation of the EPHB2 gene.

In another example, synthetic lethal screening is employed to identifygenes whose inhibition kills or slows the growth of cells that have adysfunctional or inactivated EPHB2 gene. Synthetic lethal screeningenables one to uncover a potentially novel class of drug targets ofsignificant therapeutic value. For example, two separate genes mayencode proteins that participate in a common and essential cellularfunction, where the essential nature of this function will only becomeapparent upon inactivation of both family members. Accordingly,examination of the null phenotype of each gene separately would notreveal the essential nature of the combined gene products, andconsequently, this potential drug target would not be identified.Synthetic lethality may uncover seemingly unrelated (and oftennonessential) processes, which when combined produce a synergisticgrowth impairment (e.g., cell death). In one format, synthetic lethalityis identified by using subject arrays. To achieve this, one can create“phenotype arrays” using cultured cells. Expression of each of a set ofgenes, such as the host cell's genome, can be individuallysystematically disrupted using RNA interference. Combination withalterations in oncogene and tumor suppressor pathways can be used toidentify synthetic lethal interactions that may identify noveltherapeutic targets. Other methods suitable for synthetic lethalscreening of novel drug targets include, but are not limited to, thosedescribed in U.S. Patent Application Publication No. 20040121324, whichis incorporated herein by reference. Once a novel drug target isidentified, compounds or agents can be screened for capabilities tomodulate (e.g., inhibit) the expression or protein activity of thatnovel drug target. Any screen method described herein can be used forthis purpose.

In Vivo Assays

The present invention also encompasses the use of various animal models.For instance, by developing or isolating mutant cells lines that fail toexpress normal EphB2, one can generate cancer models in mice that willbe highly predictive of cancers in humans and other mammals. Thesemodels may employ the orthotopic or systemic administration of tumorcells to mimic primary and/or metastatic cancers. Alternatively, one mayinduce cancers in animals by providing agents known to be responsiblefor certain events associated with malignant transformation or tumorprogression. Finally, transgenic or knockout animals that lack a wildtype EPHB2 gene may be utilized as models for cancer development andtreatment.

Treatment of animals with test compounds will involve the administrationof the compound, in an appropriate form, to the animal. Administrationwill be by any route the could be utilized for clinical or non-clinicalpurposes, including but not limited to oral, nasal, buccal, rectal,vaginal or topical. Alternatively, administration may be byintratracheal instillation, bronchial instillation, intradermal,subcutaneous, intramuscular, intraperitoneal or intravenous injection.Specifically contemplated are systemic intravenous injection, regionaladministration via blood or lymph supply and intratumoral injection.

Determining the effectiveness of a compound in vivo may involve avariety of different criteria. Such criteria include, but are notlimited to, survival, reduction of tumor burden or mass, arrest orslowing of tumor progression, elimination of tumors, inhibition orprevention of metastasis, increased activity level, improvement inimmune effector function or improved food intake.

Rational Drug Design

One goal of rational drug design is to produce structural analogs ofbiologically active polypeptides or compounds with which they interact(agonists, antagonists, inhibitors, binding partners, etc.). By creatingsuch analogs, it is possible to fashion drugs that are more active orstable than the natural molecules that have different susceptibility toalteration or that may affect the function of various other molecules.In one approach, one would generate a three-dimensional structure forEphB2 or a fragment thereof. This could be accomplished by x-raycrystallograph, NMR, computer modeling, or by a combination of theseapproaches. An alternative approach, “alanine scan,” involves the randomreplacement of residues throughout molecule with alanine, and theresulting affect on function determined.

It also is possible to isolate an EphB2 specific antibody, selected by afunctional assay, and then solve its crystal structure. In principle,this approach yields a pharmacore upon which subsequent drug design canbe based. It is possible to bypass protein crystallography altogether bygenerating anti-idiotypic antibodies to a functional, pharmacologicallyactive antibody. As a mirror image of a mirror image, the binding siteof anti-idiotype would be expected to be an analog of the originalantigen. The anti-idiotype could then be used to identify and isolatepeptides from banks of chemically- or biologically-produced peptides.Selected peptides would then serve as the pharmacore. Anti-idiotypes maybe generated using any method suitable for producing antibodies, usingan antibody as the antigen.

Thus, one may design drugs with improved EphB2 activity or which act asstimulators, inhibitors, agonists, antagonists of mutated or wild typeEphB2 proteins or molecules affecting or affected by EphB2 function. Byvirtue of the availability of cloned EphB2 sequences, sufficient amountsof EphB2 can be produced to perform crystallographic studies. Inaddition, knowledge of the polypeptide sequences permits computeremployed predictions of structure-function relationships.

V. KITS

All of the essential materials or reagents required for detecting orsequencing EphB2 (wild type or mutant) may be assembled together in akit. This generally will comprise preselected primers and probes (e.g.,allele specific oligonucleotide or antibody). Also included may beenzymes suitable for amplifying nucleic acids including variouspolymerases (RT, Taq, Sequenase™, etc.), deoxynucleotides and buffers toprovide the necessary reaction mixture for amplification. Such kits alsogenerally will comprise, in suitable means, distinct containers for eachindividual reagent and enzyme as well as for each primer or probe.

It should be understood that the above-described embodiments and thefollowing examples are given by way of illustration, not limitation.Various changes and modifications within the scope of the presentinvention will become apparent to those skilled in the art from thepresent description.

VI. EXAMPLES Example 1 Methods and Procedures

The EPHB2 gene was identified as a tumor-suppressor gene according tothe following methods and procedures.

Cell Lines

DU 145, LNCaP and PC-3 prostate cancer cell lines were obtained fromAmerican Type Culture Collection (ATCC; Manassas, Va.). The threenon-malignant cell lines used for normalization of the NMD microarraydata were GM11496, GM00156 and GM00038, which were obtained from CoriellCell Repository (CCR, Camden, N.J.). All cell lines were grown accordingto the distributors' instructions in 175 cm² flasks (Corning) andincubated at 37° C.

Clinical Specimens

Five primary tumors and 39 metastases were obtained from Johns HopkinsUniversity, Baltimore Md. Additionally, 28 primary prostate tumors and23 metastases were obtained from the University of Basel, Switzerland. Aset of 450 normal controls was obtained from anonymous blood donors andis commercially available (Coriell, Camden, N.J.). All clinicalspecimens used in these experiments were recruited at Johns HopkinsUniversity and University of Basel and were appropriately consented.Specimens were anonymized and randomized, and were approved for analysisat TGen by Western Institutional Review Board (WIRB).

NMD on cDNA Microarray

The hybridizations were performed on 16,000 gene cDNA microarraysprinted in the Microarray Core of the National Human Genome ResearchInstitute (NHGRI)/NIH. These cDNA microarrays were generated from cDNAclones obtained from the sequence verified IMAGE clone selection fromResearch Genetics (Invitrogen Corporation). Of these clones, 75%represent genes with functional annotation and, the remaining representESTs and hypothetical proteins. The gene description of these clones wasbased on the Unigene database, build #148 and the chromosomal baselocation of these genes was updated from University of California SantaCruz's (UCSC) Genome Browser database. This update was performed throughthe GenBank accession ID of the Image clone ID. The negative controlhousekeeping genes represent a set of 88 genes (printed 8 times each)whose expression does not vary significantly in several differenttissues. For quality control, 2 slides per lot were hybridized for spotconsistency, homogeneity and signal to noise ratios. Because of thedynamic state of the Unigene database, 12.5% of the 16,000 genes arerepresented by more than one clone in the arrays. These duplicatesrepresent a unique internal control for spot consistency andhybridization reproducibility.

Both malignant and non-malignant cell lines were treated in a similarfashion. For each cell line, half of the subconfluent cells were treatedwith 100 μg/ml Emetine Dihydrochloride Hydrate (Fluka, Buchs,Switzerland), while the remaining were untreated controls. BothEmetine-treated and untreated control cells were then incubated at 37°C. for 10 hours. After the incubation, the first time point (0 min) washarvested for both treated and untreated cells. Simultaneously,Actinomycin D (Sigma-Aldrich, St Louis, Mo.) with the finalconcentration of 5 μg/ml was added to the remaining treated anduntreated cells to stop new transcription. Time points of 10 min, 30min, 1 h, 2 h, 4 h and 8 h were harvested in both groups for most of thecell lines. Cell pellets were snap-frozen and mRNA extracted by usingFastTrack kit. (Invitrogen) according to the manufacturer'sinstructions.

For each time point, the untreated sample was hybridized against theEmetine treated equivalent. Four μg of untreated mRNA was labeled withCy5-dUTP and four μg of Emetine-treated mRNA with Cy3-dUTP (AmershamBiosciences, Piscataway, N.J.) as described. Image analysis was done byDeArray software. Average intensities of the tumor samples were dividedby the average intensities of the reference sample at each microarrayspot after background intensity subtraction. Within-slide normalizationwas performed with ratio statistics method using housekeeping genes asdescribed previously. The data were quality filtered with ratio qualitymethod, which computes a quality value for each ratio. The scale for thequality values is from zero (poor quality) to one (good quality). Allratios having quality value below 0.5 were discarded from the subsequentanalysis.

Real time Quantitative RT-PCR

Real-time quantitative RT-PCR (Q-RT-PCR) was used for validation ofoverall changes in gene expression. Cells were lysed and total RNA wasextracted using RNeasy Mini Kit method (Qiagen). Using 1 μg total RNA,cDNA was generated in 100 μl RT-PCR reaction volume by ThermoScriptRT-PCR cDNA synthesis method (Invitrogen). Using gene (EphB2, p53,hMLH1, B-actin and GAPDH) specific Assay-on-Demand TaqMan assay(PE/Applied Biosystems, Piscataway, N.J.), consisting of a specificfluorogenic probe and a pair of oligonucleotides, standard Q-RT-PCRreactions were run in an Opticon 2 real-time Q-PCR instrument (MJResearch). The reactions for the Q-RT-PCR application were carried outin a 96-well plate format with 20 μl reaction volume in triplicates. Theamount of total RNA in each TaqMan reaction was 10-50 ng. Normalizationof our data was achieved by including separate tube reactions ofreference genes GAPDH and B-actin. Individual time-points ofEmetine-treated cells were normalized relative to untreated controls inthe presence of reference genes.

CGH on cDNA Microarray

Comparative genomic hybridization (CGH) was done on 13K.Human 1 cDNAmicroarray slides from Agilent Technologies (Palo Alto, Calif.) asdescribed in Pollack, et al., NAT. GENET., 23:41-46 (1999); Hyman, etal., CANCER RES., 62:6240-6245 (2002); and Chen, et al., BIOINFORMATICS,18:1207-1215 (2002); with slight modifications. DNA obtained fromhealthy male individuals was used as a reference. Twenty μg of genomicDNA was digested overnight using AluI and RsaI (Life Technologies,Rockville, Md.). Digested DNAs were purified by phenol/chloroformextraction. Six μg of digested tumor DNA and reference DNA was labeledwith Cy5-dUTP and Cy3-dUTP (Amersham Biosciences, Piscataway, N.J.),respectively, in a random priming reaction using Bioprime Labeling kit(Life Technologies). Hybridization and washes were performed asdescribed in Hyman, et al, supra. Microarrays were scanned using a laserconfocal scanner (Agilent Technologies, Palo Alto, Calif.) and FeatureExtraction software was used to measure the fluorescence intensities atthe target locations (Agilent Technologies).

Data Analysis

A custom-made database (FileMaker Pro 5.0v3) was created including thegenomic sequence alignment information for all available mRNA sequencesaccording to the assembly by the UCSC, as well as the Unigeneinformation obtained from Build 146. The intensity ratios (i.e., NMDratio) and ratio quality values from all cDNA microarray hybridizationswere collected into the database. The intensity ratios of each prostatecancer cell line were then normalized against the average intensityratios obtained from three non-malignant cell lines (i.e. normalized NMDratio) for each clone on the array. To prioritize new candidates formutational analysis, genes with normalized NMD-ratio above three andwhose ratio quality in that cell line were above 0.5 were firstselected. Additionally, the ratios for these genes in normal controlcell lines were selected to be below 2 to filter out non-mutationinduced changes in expression. The second prioritization criterionincluded information about the deletion status of these genes, asmeasured by array CGH. To plot the CGH profiles, moving mean ratio of 30consecutive clones was used. Based on the normal variation in thecontrol hybridization, mean intensity ratios below 0.9 were consideredlosses. Based on the genomic region each gene mapped to and thecumulative copy number estimate for the clones in that loci, genes werescored as either being in a deleted region or not, as illustrated inFIG. 2.

Mutation Analysis

DNA specimens were amplified using standard PCR protocol and intronicprimer pairs with M13 tails (sequences available on request). The PCRproducts were purified using the QiaQuick PCR purification kit on theBioRobot 8000 Automated Nucleic Acid Purification and Liquid Handlingsystem (Qiagen). Quarter volume cycle sequencing reactions were preparedin 96 well format using standard M13 forward or reverse primers with theBig Dye Terminator Chemistry (PE/Applied Biosystems, Piscataway, N.J.).Following Sephadex purification, sequence products were separated on anABI 3700 or ABI 3730 Capillary DNA Analyzer (PE/Applied Biosystems,Piscataway, N.J.) using manufacturer's protocols. Sequence chromatogramswere aligned and analyzed using Sequencher version 4.1 (Gene Codes).

Colony Formation Assay

The human clone of EphB2 was purchased from OriGene Technologies andsubcloned into the pIRES-dsRed2 expression vector (BD-Biosciences). Twosubclones (a and b) were selected and validated for full-length wildtype sequence. Cells were transfected with 1.0 μg of plasmid DNA usingLipofectamine 2000 (Invitrogen) according to manufacturers instructionswith the following modification. Trypsinized cells (1.75×10⁵) wereplated with DNA-lipid complex in duplicate wells. After 24 hours, 0.5μg/mL G418 containing media was added to the wells and media changedevery two days. Fourteen days later Cell Titer Blue (Promega) reagentwas added to the wells to measure cell proliferation according tomanufacturer's instructions. Data were normalized to the vector control(pIRES-dsRed2) and presented as percentage of control proliferation.Colony formation was measured after Cell Titer Blue assay by removingmedia and fixing cells with 2% paraformaldehyde for 15 minutes. Aftertwo washes, cell colonies were stained with Giemsa stain for 30 minutes.Colonies (>1.0 mm) were visually scored by two different individualsindependently.

In addition, the TP53 cell line mutational database can be found at thewebsite “perso.curie.fr/Thierry.Soussi/p53_databaseWh.htm.” TheUniversity of California Santa Cruz's (UCSC) Genome Browser database canbe found at genome.ucsc.edu. Information on Unigene build 146 can befound at www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=unigene. Microarraydata are available at the NCBI's GEO website www.ncbi.nlm.nih.gov/geo.mRNA transcript of human EPHB2 variant I can be obtained under GenBankaccession number NM_(—)017449, and mRNA transcript of human EPHB2variant 2 can be obtained under GenBank accession number NM 004442.

Example 2 A Common Nonsense Mutation in EPHB2 is Associated withProstate Cancer Risk in African American Men with a Positive FamilyHistory

The EPHB2 gene was identified as a prostate cancer tumor suppressorgene, with somatic inactivating mutations occurring in about 10% ofsporadic tumors. With its role in maintaining normal epithelial cellarchitecture and functional data indicative of a tumor suppressor, EPHB2is an attractive candidate for a genetic risk factor for prostatecancer. This example evaluated the contribution of EPHB2 to prostatecancer susceptibility by screening the entire coding sequence of theEPHB2 gene to search for germline mutations in 72 probands from theAfrican American Hereditary Prostate Cancer (AAHPC) Study Network. Tencoding sequence variants were identified. The K1019X (3055A-→T) variantwas present in 15.3% of the AAHPC probands, although it was previouslyshown to be present in 1.7% of 231 Coriell controls. In this example, acase-control analysis of the K1019X variant using AAHPC(N=72) andAfrican American sporadic prostate cancer cases (N=183), as compared toAfrican American controls (N=329), was performed. The presence of the(T) allele significantly increased risk for prostate cancer (Odds Ratioor OR=2.44; 95% Confidence Interval or CI=1.4-4.3; Fisher's 2 sidedP=0.003). Stratified analyses revealed that the frequency of K1019X wassignificantly higher for the AAHPC probands (15.3%) as compared tohealthy African American male controls (5.2%) (OR=3.31; CI 1.48-7.41;Fisher's 2-sided P=0.008). The analysis was then adjusted for individualancestry among all subjects, in order to rule out a spurious associationdue to population stratification. The ancestry-adjusted analysisconfirmed the association (P=0.01). These data suggest that thefrequency of EphB2 K1019X, which varies significantly between AfricanAmericans and European Americans, is associated with increased risk forprostate cancer in African American men with a positive family historyand, therefore, is an important genetic risk factor for prostate cancerin the African Americans.

Prostate cancer is the most common male specific malignancy in the USand disproportionately affects African-American men, who have higherincidence and mortality rates as compared to other ethnic groups.Specifically, prostate cancer is the most common malignancy in AfricanAmerican men, representing about 40% of all cancer cases. The underlyingreasons for these disparities are not well understood, although existingevidence implicates an important genetic component. Many studies ofhereditary prostate cancer (HPC) have been reported; however, few, ifany, genes have been identified which are reproducibly associated withincreased risk for prostate cancer across different study populations,emphasizing the heterogeneous nature of this disease. Despite AfricanAmerican men having the highest incidence and mortality rates ofprostate cancer in the US, very little data are available on thegenetics of familial prostate cancer in this ethnic group. Consequently,studying the genetic contributions for prostate cancer in this high-riskpopulation will have important implications for addressing the disparityof prostate cancer in African Americans.

As demonstrated above, the gene encoding the EphB2 receptor tyrosinekinase was discovered as being completely inactivated in the DU 145 cellline using NMD inhibition to enrich for genes likely to harbormutations, in combination with array CGH. Additionally, inactivatingsomatic mutations were detected in about 10% of sporadic prostatecancers with functional data supporting a tumor suppressor role forEPHB2 in prostate cancer. EPHB2 maps to 1p36, a region associated withhereditary prostate cancer linkage. The strong genomic and functionalcharacteristics of EPHB2 along with its map position near a putative HPClocus make it a strong candidate prostate cancer susceptibility gene.This example screened the EPHB2 gene by direct sequencing for thepresence of mutations in African American hereditary prostate cancercases to determine if this gene is associated with prostate cancerpredisposition in this high-risk population. The dataset consisted of 72probands from unrelated HPC multiplex families recruited as part of theAAHPC Study Network.

Subjects and Methods

African American Hereditary Prostate Cancer Cases from AAHPC Study

Ascertainment of multiplex prostate cancer families and the clinicaldescription of the AAHPC cases have been previously described (Royal, etal., ANN. EPIDEMIOL., 10:568-577 (2000); and Powell, et al., J. NATL.MED. Assoc., 93:25 S-28S (2001)). The AAHPC Study Network developed anation-wide effort to establish Collaborative Recruitment Centers (CRes)in regions of the US with large African American populations. Inclusioncriteria were: (1) four or more prostate cancer cases, preferably firstdegree relatives, (2) at least three cases available for sampling, and(3) an average age at diagnosis of <65 years of age for the family.These families all self identified as African American and were verifiedby the recruitment staff. To date 83 families fitting these criteriahave been recruited into the AAHPC study. We performed mutationalanalysis for the EPHB2 gene using DNA samples from 72 probands fromunrelated AAHPC families. The average age at diagnosis for theseprobands was 64.9 years of age. All participants gave informed consent,and recruitment was approved by the appropriate Institutional ReviewBoards (IRB).

African American Sporadic Prostate Cancer Cases and Controls

Unrelated men self-described as African American were enrolled forcase-control studies of risk factors for prostate cancer. The subjectsconsisted of 512 African Americans (183 prostate cancer patients and 329healthy male controls) recruited from the Howard University Hospital(HUH) in Washington, D.C. Incident cases were identified through theDivision of Urology at HUH and confirmed by review of medical records.Healthy control subjects unrelated to the cases and matched for age (±5years) were ascertained from the Division of Urology at HUH and alsofrom men participating in screening programs for prostate cancer at theHUH. The demographic characteristics of participants in the screeningprogram were similar to the patient population seen in the Division ofUrology clinics. Recruitment of sporadic prostate cancer cases andhealthy controls occurred concurrently, and they each donated a bloodsample for research purposes. The participation response rates for casesand controls were 92% and 90%, respectively. All prostate cancer caseswere between 40 to 85 years of age and were diagnosed with the diseasewithin the last 4 years. Clinical characteristics including Gleasongrade, prostatic specific antigen (PSA), tumor-node-metastasis (TNM)stage, age at diagnosis, and family history were obtained for all casesfrom medical records. Disease aggressiveness was defined as “Low” (Tcategory<T2c and/or Gleason grade<7) or “High” (T category>T2c and/orGleason grade>7). All healthy controls had PSA levels<4.0 ng/ml andnormal digital rectal examination (DRE). The Howard University IRBapproved the study, and written consent was obtained from all subjects.In addition, we used previously published data from 231 nonethnicallydefined population control genomic DNA samples commercially availablefrom the Coriell Institute for Medical Research in order to compare theEphB2 K1019X allele and genotype frequencies.

Mutation Detection and Genotyping

DNA specimens were amplified using standard PCR protocol and intronicprimer pairs with M13 tails. The PCR products were purified using theQiaQuick PCR purification kit on the BioRobot 8000 Automated NucleicAcid Purification and Liquid Handling system (Qiagen). Quarter or eighthvolume cycle sequencing reactions were prepared in 96 well format usingstandard M13 forward or reverse primers with the Big Dye TerminatorChemistry (PE/Applied Biosystems, Piscataway, N.J.). Following Sephadexpurification, sequence products were separated on an ABI 3700 or ABI3730 Capillary DNA Analyzer (PE/Applied Biosystems, Piscataway, N.J.)using manufacturer's protocols. Sequence chromatograms were aligned andanalyzed using Sequencher version 4.1 (Gene Codes).

Controlling for Population Stratification

To control for possible confounding by population stratification in thisstudy, a panel of 34 ancestry informative markers (AIMs) was alsogenotyped in the African American samples. These markers show largedifferences in frequency between the parental populations (West Africansand Europeans), and were used to control for the presence of populationstratification (PS) due to admixture. Information regarding primersequences, polymorphic sites and other relevant information on the AIMscan be found at the dbSNP NCBI database site, under the submitter handlePSU-ANTH (www.ncbi.nlm.nih.gov/SNP/).

Statistical Analysis

Odds ratios (ORs) and P values were determined by logistic regressionanalyses from comparison of genotypes between subjects with prostatecancer and healthy controls using SAS version 6.91 (SAS Institute, Inc,Cary, N.C.). Further analyses were performed on the combined datasetconsisting of all prostate cancer subjects, and for the hereditary andsporadic cases separately. For all analyses, genetic effects wereadjusted for age (at time of diagnosis for case subjects and at time ofascertainment for controls). Statistical control of PS was achieved byintroducing individual ancestry (IA) as a covariate in the analyses.Individual ancestry was estimated by two independent methods: themaximum likelihood approach described by Hanis, et al., AM J PHYSANTRHOPOL, 70:433-441 (1986) and a Bayesian method implemented in theSTRUCTURE 2.0 program (Pritchard, et al., GENETICS, 155:945-959 (2000)),and the estimate was then used as a covariate in the regressionanalyses.

Results

The clinical characteristics of the 72 AAHPC probands, the 183 sporadicprostate cancer cases and the 329 healthy African American male controlsare presented in Table 3. The mean age of 69 years for the sporadicprostate cancer cases was higher than that for the controls (66.1 years)and the HPC probands (64.9 years). The mean PSA for the controls wasbelow 4.0 ng/ml as expected. The Wilcox Sign-Rank test showed that themean PSA for both the HPC probands and the sporadic prostate cancercases compared with African American male controls was significantlydifferent (P value<0.01). For 52 HPC probands on whom diseaseaggressiveness categorization was available, only 17% had a high indexcompared with the 47% of those with sporadic disease.

TABLE 3 Clinical Characteristics of African American Prostate Cancer(PC) Patients and Population-Based Control Subjects Hereditary PCSporadic PC Controls Characteristic (N = 72) (N = 183) (N = 329) Meanage in years (SD) 64.9 (20.8) 69.0 (8.9)  66.1 (12.6) Mean serum PSA in 52.3 (90.1)^(b)   71.3 (195.1)^(b) 2.8 (1.1) ng/ml (SD)^(a) DiseaseAggressiveness:^(c) Low (%) 43 (83) 54 (53) — High (%)  9 (17) 48 (47) —Unknown 20 81 — ^(a)Serum PSA measured at time of diagnosis for casesand at most recent clinical visit for controls. ^(b)P-value <0.01 fromWilcoxon Sign-Rank test comparison with control population. ^(c)Lowaggressiveness: Gleason <7 and T category <T2c; High aggressiveness:Gleason ≧7 or T category ≧T2c.

Mutational analysis in 72 AAHPC probands resulted in the discovery often unique coding sequence variants within the EPHB2 gene (Table 4).Only four of these variants actually resulted in amino acid changes andare considered mutations (Table 4). Included among these codingmutations is the previously reported K1019X nonsense mutation (3055A→T)in exon 15 of the EPHB2 gene. This K1019X mutation was present in 15.3%(11 of 72) of the AAHPC probands, though it was previously shown to bepresent in 1.7% (4 of 231) of control DNA samples from the CoriellInstitute for Medical Research (Odds Ratio or OR=10.23; 95% CI3.15-33.26; two-sided Fisher's exact test P-value of 0.000043). Themutation was present in 5.17% of the African American controls (17 of329) and is therefore three times more common among African Americansthan European Americans, suggesting that it may be in admixturedisequilibrium in the African American population.

TABLE 4 Characterization of 10 Coding Variants Discovered within EPHB2in 72 AAHPC Probands Amino Acid Frequency in Nucleotide positionConsequence dbSNP Probands  1. 51OC→T none — 5.6%  2. 624G→A none — 1.4% 3. 657G→A none rs1371869 1.4%  4. 835G→T A279S — 2.8%  5. 930C→T none —5.6%  6. 1377G→A none rs2229872  30%  7. 1949T→C V650A — 2.8%  8.2640G→A none — 1.4%  9. 2647A→G M883V — 2.8% 10. 3055A→T K1019X — 15.3% ^(a)Nucleotide and amino acid positions are based upon coding sequencefor GenBank accession file NM_017449. Specifically, the nucleotidenumbering is with reference to nucleotide 19 of SEQ ID NO: 2, and theamino acid numbering is with reference to amino acid #1 of SEQ ID NO: 1.

An association analysis of the K1019X variant (3055A→T) was performedcombining all AAHPC(N=72) and sporadic cases (N=183) and comparing themto African American male controls (N=329) controlling for age atdiagnosis (Table 5). The presence of the (T) allele significantlyincreased risk for prostate cancer (OR=2.44; 95% CI=1.4-4.3; Fisher's 2sided P=0.003). Stratified analyses (Table 5) revealed that thefrequency of K1019X was significantly higher for the AAHPC probands(15.3%) as compared to African American healthy male controls (5.2%)(OR=3.31; CI 1.48-7.41; Fisher's 2-sided P=0.008). The 6.6% (12 of 183)frequency of the mutation among the 183 sporadic prostate cancer caseswas compared with the African American healthy male controls, and foundno significant difference between the two groups (OR, 1.28; 95% CI,0.6-2.8; Fisher's 2-sided P=0.55)

TABLE 5 Association between Prostate Cancer and EphB2 KI019XPolymorphism No. (%) of subjects with Population N K/K K/X X/X OR (95%CI)^(a) P-value All Cases 255 234 (91.7) 19 (7.5)  2 (0.7) 2.44(1.4-4.3) 0.003 AAHPC 72  61 (84.7) 11 (15.3) — 3.31 (1.5-7.4) 0.008 AAsporadic PC^(b) 183 173 (94.5) 8 (4.4) 2 (1.1) 1.28 (0.6-2.8) 0.55  AAcontrols 329 312 (94.8) 9 (3.2) 8 (1.9) 1.00 (reference) — Coriellcontrols 231 270 (98.5) 4 (1.5) — ^(a)OR by logistic regression for KJXand XIX genotype comparisons with African American controls afteradjusting for age at diagnosis. ^(b)AA denotes African American, and PCstands for prostate cancer

The K1019X variant was not in Hardy Weinberg Equilibrium (HWE) withinthe African American sporadic cases and control samples (p<0.05). Theobserved departure from HWE was not unexpected given that the AfricanAmerican population is the product of recent admixture and there weresignificant differences in K1019X allele frequency between AfricanAmericans and European Americans. Thus, in order to rule out a spuriousassociation of K1019X with prostate cancer in African Americans due toadmixture stratification, the analysis was controlled for ancestraldifferences between the African American cases and controls byestimating individual ancestry for each subject using 34 admixtureinformative markers (AIMs). The individual ancestry (IA) estimate foreach subject was used as a covariate in the association analysis inorder to take into account differences in ancestral proportions betweencases and controls. Individual ancestry (West African) ranged from 10%to 93.5% in the cases with an average IA estimate of 71.3±1.9. Theestimates for West African ancestry for the controls ranged from 6.5% to95.3% (average value was 6.9.0±0.8). After adjusting for individualancestry, the association of the EphB2 K1019X mutation with prostatecancer in the AAHPC probands as compared to the African American healthymale controls was still significant (P=0.01).

Finally, all sampled family members of 11 mutation positive probandswere screened to test whether or not the K1019X mutation tracks withprostate cancer in these families. Of the 11 families with mutationpositive probands, the mutation was present in at least two of threecases in six of these families. In one family the K1019X mutation waspresent in three of four affected brothers. DNA was not available forthe fourth affected brother. While these data do not show completetransmission of the mutation with prostate cancer, partial transmissionof the mutation is evident in multiple families.

Discussion

This example investigated the potential genetic basis for the highincidence and mortality rates of prostate cancer among African Americansand examined the association of the EPHB2 gene and prostate cancer riskin African Americans. The EPHB2 gene was demonstrated to be a prostatecancer tumor suppressor gene. Somatic inactivating mutations in thisgene were discovered in the DU 145 prostate cancer cell line and inclinical prostate tumor samples. Further evidence for its role as atumor suppressor gene were also reported, where wild type EphB2significantly reduced clonogenic growth of DU 145 prostate cancer cells,which have biallelic inactivation of EphB2. This example identified tensequence variants in the EPHB2 gene among 72 African American hereditaryprostate cancer patients including the K1019X nonsense mutation. TheK1019X variant was observed in much higher frequency among AfricanAmerican prostate cancer patients than among healthy African Americanmale controls (P=0.003). The association was mainly due to men withhereditary prostate cancer (P=0.008). In fact the risk for prostatecancer was increased 3-fold among African American men who carried atleast one copy of the K1019X allele and had a family history of prostatecancer. This high frequency of this mutation in hereditary casessuggests it is likely to be associated with familial prostate cancer inAfrican American men.

The prevalence of K1019X was also significantly higher among AfricanAmerican controls than among European American controls (P<0.001),suggesting that it may be in admixture disequilibrium in the AfricanAmerican population. These findings inspire further investigation on therole of this mutation as a prostate cancer genetic risk factor; however,several questions remained. Ethnic differences in allele frequency anddisease risk can create false-positive results in case-control studies,especially when using recently admixed populations such as AfricanAmericans. Thus, in order to control for possible confounding, thisexample introduced individual ancestry as a covariate in the analyses.This approach has been used to limit spurious associations that are theresult of differences in ancestral proportions (admixture). Theancestry-adjusted analyses provided additional support for a strongassociation of the K1019X and prostate cancer in African Americans.

The frequencies of sequence variants in a number of candidate genes forprostate cancer differ significantly between African Americans andEuropean Americans. Among the examples are the CAG repeat tract withinthe androgen receptor gene, a TA-repeat tract within the SRD5AR gene,the CYP3A4 promoter variant and frequent variants within MSRI. The EphB2K1019X mutation represents a novel addition to this group of allelicvariants.

The examination of sequence variants in the EPHB2 gene and subsequentcase control study among African American men suggest that EPHB2 has animportant role in familial prostate cancer. This finding is significantgiven the higher frequency of the EphB2 K1019X nonsense mutation and thehigher prevalence of prostate cancer among African American men comparedto their US counterparts.

The foregoing description of the present invention provides illustrationand description, but is not intended to be exhaustive or to limit theinvention to the precise one disclosed. Modifications and variations arepossible consistent with the above teachings or may be acquired frompractice of the invention. Thus, it is noted that the scope of theinvention is defined by the claims and their equivalents.

1. A method of selecting a pharmaceutical composition capable ofcorrecting the phenotype resulting from a change in expression orfunction caused by a 3055A→T mutation in a polynucleotide comprising anEPHB2 gene (SEQ ID NO.: 2 or 4) comprising: culturing a cell comprisinga 3055A→T mutation in a polynucleotide comprising an EPHB2 gene (SEQ IDNO.: 2 or 4) contacting a candidate pharmaceutical composition with thecell evaluating the capability of the candidate pharmaceuticalcomposition to correct the result.
 2. The method of claim 1 wherein thecandidate pharmaceutical composition comprises a chemical compound. 3.The method of claim 1 wherein the candidate pharmaceutical compositioncomprises an RNAi.
 4. The method of claim 1 wherein the candidatepharmaceutical composition comprises an antibody Fv region.
 5. Themethod of claim 1 wherein the phenotype comprises a change in theexpression of a gene other than the EPHB2 gene (SEQ ID NO.: 2 or 4) 6.The method of claim 5 wherein the change in the expression of a geneother than EPHB2 is measured by the detection of a change in mRNAexpression.
 7. The method of claim 5 wherein the change in theexpression of a gene other than EPHB2 is measured by the detection of achange in protein expression.
 8. The method of claim 1 wherein thephenotype comprises abnormal cell growth.
 9. The method of claim 8wherein the candidate pharmaceutical composition is capable ofconditionally suppressing the abnormal cell growth.
 10. The method ofclaim 1 wherein the cell is derived from a prostate cancer.
 11. Themethod of claim 10 wherein the cell is derived from an African-Americanman.
 12. The method of claim 43 wherein the culturing occurs within ananimal.